CN109071561B - Thienopyrazine carboxamides as ubiquitin-specific protease inhibitors - Google Patents

Abstract

The present disclosure relates to inhibitors of USP28 and/or USP25 useful for treating cancer, inflammation, autoimmune and infectious diseases, the inhibitors having formula (I):

wherein R is₁、R₂、R₃、R₄、R_4'、R₅、R₆X and n are as described herein.

Description

Thienopyrazine carboxamides as ubiquitin-specific protease inhibitors

RELATED APPLICATIONS

This application claims the benefit and priority of U.S. provisional application No. 62/294,583 filed on 12.2.2016, the entire contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present disclosure relates to modulators of ubiquitin-specific protease 28(USP28) and/or ubiquitin-specific protease 25(USP25) which are useful for treating diseases or disorders associated with the USP28 and/or USP25 enzymes. In particular, the disclosure relates to compounds and compositions that inhibit USP28 and/or USP25, methods of treating diseases or disorders associated with USP28 and/or USP25, and methods of synthesizing these compounds.

Background

USP28 and USP25 are cysteine isopeptidases of the USP subfamily of DUBs containing the following three distinct domains: an N-terminal UBA-like domain; a pair of Ubiquitin Interacting Motifs (UIM) and a USP domain predicted to have a conserved fold of the USP subfamily (Nijman et al, Cell 2005, 123, 773-42 786; Komanter et al, mol. Cell Bio.2009, 10, 550-563). USP28 and USP25 exert their functions by modulating the stability of hypercellular proteins. USP28 has been characterized as a tumor promoting factor and has been found to stabilize a number of oncoproteins. USP25 has been characterized as a tumor promoting factor and as a modulator of cellular responses associated with autoimmune, inflammatory and infectious diseases such as viruses and bacteria.

Amplification, deletion and mutation of USP28 have been identified in a variety of cancer types, including breast, AML, ovarian and colorectal cancers. (cbioportal; http:// www.cbioportal.org; Diefenbacher et al, J.of Clin.investi.2014, 124, 3407-. Furthermore, USP28 overexpression correlates with a poor prognosis in patients with glioblastoma, non-small cell lung cancer and bladder cancer, suggesting that USP28 plays an important role in tumorigenesis of these tumor types. (Wang et al Exp. biol. Med.2016, 255-.

Large-scale shRNA screening also identified a role for USP28in controlling stability of MYC proteins. (Popov, nat. cell. biol., 765. cozone 774). MYC is a major regulator of gene transcription involved in cell growth, proliferation and apoptosis, and is critical for tumor initiation and maintenance in many tumor types. (Meyer et al, nat. Rev. cancer 2008, 8, 976. sup. 990; Conacci-Sorrell et al, Cold Spring Harb. Perspect. Med.2014, 4, 1-24; Huang et al, Cold Spring Harb. Perspect. Med. 2013; Roussel et al, Cold Spring Harb. Perspect. Med. 2013; Gabay et al, Cold Spring Harb. Perspect. Med. 2014; Schmitz et al, Cold Spring Harb. Perspect. Med. 2014.). Moreover, MYC is the most commonly amplified oncogene in human cancers, altered in many tumor types including breast, lung and prostate cancers. (Beroukhim et al, Nature 2010, 463, 899-. Knock-down of the USP28 gene has been shown to result in a reduction of MYC proteins and associated growth inhibition in a panel of human cancer cell lines in vitro. (Popov, nat. cell biol., 765-774).

USP28 has also been reported to be essential for conferring protein stability to LSD1 (lysine-specific demethylase 1). (Wu et al, Cell Rep.2013, 5, 224- & 236). LSD1 is a histone demethylase that complexes with many chaperones to control cell pluripotency and differentiation. (Metzger et al Nature 2005, 437, 436-. It has been shown that the knockdown of USP28in tumor cells results in destabilization of the LSD1 protein, inhibition of Cancer Stem Cell (CSC) -like features in vitro, and inhibition of tumor growth in vivo. (Wu, Cell Rep., 224-. Small molecule inhibitors of LSD1 showed antitumor activity in AML and ewing sarcoma models. (Sankar et al, "Reversible LSD1 inhibition interactions with global EWS/ETS translational activity and impresses with a atomic growth," Clin Cancer Res. 20144584-4597; Schenk et al, nat. Med.2012, 18, 605-. Thus, USP28 inhibition represents an alternative approach to targeting LSD1 in these tumor types.

USP28 inhibition has also been shown to reduce NICD1 levels and result in inhibition of NOTCH pathway activity. (Diefenbacher et al.). NOTCH signaling controls different cellular differentiation decisions and drives tumorigenesis in certain tumor types. NOTCH1 is a potent T cell oncogene, with > 50% of T cell acute lymphoblastic leukemia (T-ALL) cases carrying activating mutations in NOTCH hl. (Weng et al Science 2004, 306, 269-271). Increased NOTCH1 protein levels are also associated with disease progression in colon cancer. (Meng et al, Cancer Res.2009, 69, 573-. NOTCH1 rearrangement leads to constitutive pathway activation and drives tumorigenesis in many cancer types including triple negative breast cancer. (Stoeck et al, Cancer Discov.2014, 4, 1154-.

Other reported substrates of USP28 include c-Jun, Cyclin E, HIF-1 α, Claspin, 53BP1 and Mdcl, many of which play an important role in human tumorigenesis. (Diefenbacher et al; Fligel et al, blood 2012, 119, 1292-. Interestingly, the substrate recognition subunit of SCF (FBW7) E3 ubiquitin ligase, FBW7, recognizes many USP28 substrates. (Diefenbacher et al.). FBW7 recognizes USP28 substrates in a phosphorylation-dependent manner and targets them for ubiquitination, ultimately leading to their proteasomal degradation. The antagonism of its shared oncoprotein substrate by USP28 and FBW7 suggests a complex nature of protein stability control and may provide additional therapeutic opportunities for cancer treatment.

USP28 germ line knockout mice have been shown to be viable and fertile, confirming that USP28 activity is not required for normal development and reproductive function. (Knobel et al, Molecular and Cellular Biology 2014, 34, 2062-2074). Conditional knockdown of USP28in the intestine of mice resulted in a reduction in oncoproteins, including c-Myc, active NOTCH (NICD1) and c-JUN, which are associated with reduced intestinal cell proliferation and enhanced differentiation. More importantly, intestinal tumorigenesis induced by APC mutation was effectively blocked by exhaustion of acute USP28, suggesting that USP28 may be an attractive target for reducing tumor burden and improving survival in intestinal cancers. (Diefenbacher et al.).

In summary, USP28 and USP25 play important roles in promoting tumorigenesis in cells and modulating immune responses. Its main role is to de-ubiquitinate and stabilize a variety of oncoproteins and epigenetic and immunoregulatory proteins and other cytokines, which are essential for human immune response and tumor initiation and growth. Thus, inhibition of USP28 with small molecule inhibitors has the potential to treat cancer, autoimmune diseases, inflammatory diseases, infectious diseases and other disorders. To this end, there remains a considerable need for novel and effective small molecule inhibitors of USP28 and/or USP 25.

Summary of The Invention

A first aspect of the present disclosure relates to a compound of formula (I):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof,

wherein:

x is N or CR₆；

R₁Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -CN, or-NR₈R₉；

R₂Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁；

Or R₁And R₂Together form optionally substituted by one or more R₁₂Substituted (C)₄-C₈) A cycloalkyl group;

R₃is H, (C)₁-C₆) Alkyl, or (C)₁-C₆) A haloalkyl group;

R₄is H, (C)₁-C₆) Alkyl, halogen, or (C)₁-C₆) A haloalkyl group;

R_4′is H, (C)₁-C₆) Alkyl, halogen, or (C)₁-C₆) A haloalkyl group;

R₅is- (C)₀-C₃) alkylene-C (O) OH, - (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, - (C)₀-C₃) Alkylene-aryl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, wherein heterocycloalkyl, aryl and heteroaryl are optionally substituted by one or more R₁₃Substitution;

each R₆Independently at each occurrence is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, halogen, -H, -CN, (C)₃-C₈) Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl is optionally substituted with one or more (C)₁-C₆) Alkoxy or-OH, and wherein the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one or more R₁₄Substitution; or

R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅Substituted (C)₄-C₈) A cycloalkyl ring; or R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅A substituted heterocycloalkyl ring; r₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅A substituted aryl ring; or R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅A substituted heteroaryl ring; or

Two R₆When on adjacent atoms form together (C)₄-C₈) A cycloalkyl ring; or two R₆When on adjacent atoms, together form a heterocycloalkyl ring; two R₆When on adjacent atoms together form an aryl ring; or two R₆When on adjacent atoms, together form a heteroaryl ring;

R₇is H or (C)₁-C₆) An alkyl group;

each R₈、R₉、R₁₀And R₁₁Independently H, (C)₁-C₆) Alkyl, or-C (O) (C)₁-C₆) An alkyl group;

each R₁₂Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, halogen, or-OH;

each R₁₃Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, - (O) NR₁₈R₁₉、 -S(O)₂(C₁-C₆) Alkyl, -OH or-NR₁₆R₁₇Wherein the alkyl is optionally substituted with one or more substituents independently selected from: (C)₁-C₆) Alkoxy, OH and heterocycloalkyl; or

When R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together may form-C ═ (O) when attached to the same carbon; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spiro cycloalkyl; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spiroheterocycloalkyl; or two R₁₃When on adjacent atoms together form an optionally substituted one or more R₂₀A substituted heterocycloalkyl ring; or two R₁₃When on adjacent atoms together form an optionally substituted one or more R₂₀A substituted heteroaryl ring; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together with the atoms to which they are attached may form optionally substituted by one or more R₂₀A substituted bridged heterocycloalkyl ring;

each R₁₄Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, halogen, cycloalkyl, heterocycloalkyl, or-C (O) -heterocycloalkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from (C)₁-C₆) Alkoxy and-OH;

each R₁₅Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, halogen, -OH, -CN, -C (O) OH, or-C (O) O (C)₁-C₆) An alkyl group;

each R₁₆And R₁₇Independently H, (C)₁-C₆) Alkyl, (C)₃-C₈) Cycloalkyl, -CH₂C(O)NH₂、 -S(O)₂(C₁-C₆) Alkyl, -S (O)₂(C₆-C₁₀) Aryl or-C (O) (C)₁-C₆) An alkyl group;

each R₁₈And R₁₉Independently is H or (C)₁-C₆) An alkyl group;

each R₂₀Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, or halogen; or

Two R₂₀Together form-C ═ (O) when attached to the same carbon; and is

n is 0, 1, 2, or 3.

Another aspect of the disclosure relates to methods of treating a disease or disorder associated with inhibition of USP 28. The method comprises administering to a patient in need of treatment of a disease or disorder associated with inhibition of USP28 an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

Another aspect of the disclosure relates to methods of treating a disease or disorder associated with inhibition of USP 25. The method comprises administering to a patient in need of treatment of a disease or disorder associated with inhibition of USP28 an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

Another aspect of the present disclosure relates to methods of treating diseases or disorders associated with inhibition of USP28 and USP 25. The method comprises administering to a patient in need of treatment of a disease or disorder associated with inhibition of USP28 an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

Another aspect of the disclosure relates to a method of inhibiting USP 28. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to a method of inhibiting USP 25. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present disclosure relates to methods of inhibiting USP28 and USP 25. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to a method of treating cancer. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to a method of treating inflammation. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to methods of treating autoimmune diseases. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to methods of treating infectious diseases. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to a method of treating a viral infection. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the disclosure relates to a method of treating a bacterial infection. The method comprises administering to a patient in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present disclosure relates to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise an excipient, diluent or surfactant.

Another aspect of the present disclosure relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the manufacture of a medicament for the treatment of a disease associated with the inhibition of USP 28.

Another aspect of the present disclosure relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the treatment of a disease associated with inhibition of USP 28.

Another aspect of the present disclosure relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the treatment of a disease associated with inhibition of USP 25.

Another aspect of the present disclosure relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the treatment of a disease associated with inhibition of USP28 and USP 25.

The present disclosure further provides methods of treating diseases or disorders associated with modulation of USP28 and/or USP25, including cancer, inflammation, autoimmune diseases, viral infections and bacterial infections, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof to a patient suffering from at least one of the diseases or disorders.

The present disclosure provides inhibitors of USP28 and/or USP25 which are therapeutic agents in the treatment of diseases such as cancer, inflammation, autoimmune diseases, viral infections and bacterial infections. Finally, the present disclosure provides the medical community with novel pharmacological strategies for treating diseases and disorders associated with the USP28 and/or USP25 enzymes.

Detailed Description

The present disclosure relates to compounds and compositions capable of inhibiting USP28 and/or USP25 activity. The disclosure features methods of treating, preventing, or alleviating a disease or disorder in which USP28 and/or USP25 plays a role by administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. The methods of the present disclosure can be used to treat a variety of USP28 and/or USP25 dependent diseases and disorders by inhibiting the activity of USP28 and/or USP25 enzymes. Inhibition of USP28 and/or USP25 provides novel methods of treating, preventing or ameliorating diseases, including but not limited to cancer.

In a first aspect of the disclosure, compounds of formula (I) are described:

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof, wherein R is₁、R₂、R₃、R₄、R_4′、R₅、R₆X and n are as described herein above.

The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are herein incorporated by reference in their entirety.

Definition of

The articles "a" and "an" as used in this disclosure refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "and/or" as used in this disclosure means "and" or "unless otherwise indicated.

The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., an alkyl group) may be (but is not required to be) bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (e.g., pure hydrocarbon). Alternatively, the same optionally substituted alkyl group may have a substituent other than hydrogen. For example, it may be bonded to a halogen atom, a hydroxyl group, or any other substituent described herein at any position in the chain. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but not necessarily any additional functional groups. Suitable substituents for use in the optional substitution of the groups include, but are not limited to, halogen, oxo, -H, -CN, -COOH, -CH₂CN、-O-(C₁-C₆) Alkyl, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, -O- (C)₂-C₆) Alkenyl, -O- (C)₂-C₆) Alkynyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, -OH, -OP (O) (OH)₂、-OC(O)(C₁-C₆) Alkyl, -C (O) (C)₁-C₆) Alkyl, -OC (O) O (C)₁-C₆) Alkyl, -NH₂、-H((C₁-C₆) Alkyl), ((C)₁-C₆) Alkyl radical)₂、-NHC(O)(C₁-C₆) Alkyl, -C (O) NH (C)₁-C₆) Alkyl, -S (O)₂(C₁-C₆) Alkyl, -S (O) NH (C)₁-C₆) Alkyl, and S (O) N ((C)₁-C₆) Alkyl radical)₂. The substituents themselves may be optionally substituted. As used herein, "optionally substituted" also refers to substituted or unsubstituted, the meaning of which is described below.

As used herein, the term "substituted" means that a particular group or moiety has one or more suitable substituents, wherein the substituents may be attached to the particular group or moiety at one or more positions. For example, aryl substituted with cycloalkyl may represent a cycloalkyl group attached to one atom of an aryl group by a bond or by being fused to an aryl group and sharing two or more common atoms.

As used herein, the term "unsubstituted" means that the specified group has no substituents.

Unless otherwise specifically defined, the term "aryl" refers to a cyclic aromatic hydrocarbon group having 1 to 3 aromatic rings, including monocyclic or bicyclic groups, such as phenyl, biphenyl, or naphthyl. In the case of containing two aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group can be connected at a single point (e.g., biphenyl), or fused (e.g., naphthyl). The aryl group may be optionally substituted at any point of attachment with one or more substituents (e.g., 1 to 5 substituents). Exemplary getSubstituents include, but are not limited to: -H, -halogen, -O- (C)₁-C₆) Alkyl, (C)₁-C₆) Alkyl, -O- (C)₂-C₆) Alkenyl, -O- (C)₂-C₆) Alkynyl, (C)₂-C₆) Alkenyl, (C)₂-C₆) Alkynyl, -OH, -OP (O) (OH)₂、-OC(O)(C₁-C₆) Alkyl, -C (O) (C)₁-C₆) Alkyl, -OC (O) O (C)₁-C₆) Alkyl, -NH₂、NH((C₁-C₆) Alkyl), N ((C)₁-C₆) Alkyl radical)₂、 -S(O)₂-(C₁-C₆) Alkyl, -S (O) NH (C)₁-C₆) Alkyl, and-S (O) N ((C)₁-C₆) Alkyl radical)₂. The substituents themselves may be optionally substituted. Further, when containing two fused rings, an aryl group as defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems for these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthyl, tetrahydrobenzendienyl, and the like.

Unless otherwise specifically defined, "heteroaryl" means a monovalent monocyclic or polycyclic aromatic group of 5 to 24 ring atoms containing one or more ring heteroatoms selected from N, O and S, the remaining ring atoms being C. Heteroaryl as defined herein also denotes a bicyclic heteroaromatic group wherein the heteroatom is selected from N, O and S. The aromatic groups are independently optionally substituted with one or more substituents described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolinyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno [3, 2-b ] thiophene]Thiophene, triazolyl, triazinyl, imidazo [1, 2-b ]]Pyrazolyl, furo [2, 3-c ] s]Pyridyl, imidazo [1, 2-a ]]Pyridyl, indazolyl, pyrrolo [2, 3-c ]]Pyridyl, pyrrolesAnd [3, 2-c ]]Pyridyl, pyrazolo [3, 4-c]Pyridyl, thieno [3, 2-c]Pyridyl, thieno [2, 3-c ]]Pyridyl, thieno [2,3-b ]]Pyridyl, benzothiazolyl, indolyl, indolinyl, indolinonyl, dihydrobenzothienyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxazinyl, quinolinyl, isoquinolinyl, 1, 6-naphthyridinyl, benzo [ de ] de]Isoquinolinyl, pyrido [4, 3-b ]][1，6]Naphthyridinyl, thieno [2,3-b ]]Pyrazinyl, quinazolinyl, tetrazolo [1, 5-a ]]Pyridyl, [1, 2, 4 ] or a salt thereof]Triazolo [4, 3-a]Pyridyl, isoindolyl, pyrrolo [2,3-b ]]Pyridyl, pyrrolo [3, 4-b]Pyridyl, pyrrolo [3, 2-b]Pyridyl, imidazo [5, 4-b ]]Pyridyl, pyrrolo [1, 2-a ]]Pyrimidinyl, tetrahydropyrrolo [1, 2-a ] s]Pyrimidinyl, 3, 4-dihydro-2H-1 Lambda²-pyrrolo [2, 1-b]Pyrimidine, dibenzo [ b, d ]]Thiophene, pyridine-2-ones, furo [3, 2-c ]]Pyridyl, furo [2, 3-c ]]Pyridyl, 1H-pyrido [3, 4-b ]][1，4]Thiazinyl, benzoxazolyl, benzisoxazolyl, furo [2,3-b]Pyridyl, benzothiophenyl, 1, 5-naphthyridinyl, furo [3, 2-b ] and their use as pesticides]Pyridine, [1, 2, 4 ]]Triazolo [1, 5-a]Pyridyl, benzo [1, 2, 3] s]Triazolyl, imidazo [1, 2-a ]]Pyrimidinyl, [1, 2, 4 ] or their salts]Triazolo [4, 3-b]Pyridazinyl, benzo [ c)][1，2，5]Thiadiazolyl, benzo [ c ]][1，2，5]Oxadiazole, 1, 3-dihydro-2H-benzo [ d]Imidazol-2-one, 3, 4-dihydro-2H-pyrazolo [1, 5-b][1，2]Oxazinyl, 4,5, 6, 7-tetrahydropyrazolo [1, 5-a]Pyridyl, thiazolo [5, 4-d ]]Thiazolyl, imidazo [2, 1-b ]][1，3，4]Thiadiazolyl, thieno [2,3-b ]]Pyrrolyl, 3H-indolyl and derivatives thereof. Further, when containing two fused rings, heteroaryl groups as defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3, 4-dihydro-1H-isoquinolinyl, 2, 3-dihydrobenzofuran, indolinylIndolyl and dihydrobenzoxazinyl.

Halogen or "halo" refers to fluorine, chlorine, bromine or iodine.

Alkyl refers to straight or branched chain saturated hydrocarbons containing 1 to 12 carbon atoms. (C)₁-C₆) Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

"alkoxy" refers to a straight or branched chain saturated hydrocarbon containing from 1 to 12 carbon atoms containing a terminal "O" in the chain, such as-O (alkyl). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, tert-butoxy or pentoxy groups.

The term "alkylene (or alkylenyl)" refers to a divalent alkyl group. Any of the above monovalent alkyl groups can be an alkylene group by abstraction of a second hydrogen atom from the alkyl group. Alkylene may also be C, as defined herein₀-C₆An alkylene group. Alkylene may further be C₀-C₄An alkylene group. Typical alkylene groups include, but are not limited to, -CH₂-、-CH(CH₃)-、-C(CH₃)₂-、-CH₂CH₂-、-CH₂CH(CH₃)-、 -CH₂C(CH₃)₂-、-CH₂CH₂CH₂-、-CH₂CH₂CH₂CH₂-and the like.

"cycloalkyl" or "carbocyclyl" means a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl (norbomanyl), norbornenyl (norbomenyl), bicyclo [2.2.2]Octyl, or bicyclo [2.2.2]Octenyl and derivatives thereof. C₃-C₈Cycloalkyl is a cycloalkyl group containing between 3 and 8 carbon atoms. Cycloalkyl groups may be fused (e.g., decalin) or bridged (e.g., norbornane).

"Heterocyclyl" or "heterocycloalkyl" rings contain carbon and heteroatoms selected from oxygen, nitrogen or sulfur, and in which there is no delocalized π -atom (aromaticity) between ring carbons or heteroatoms. The heterocycloalkyl ring structure may be substituted with one or more substituents. The substituents themselves may be optionally substituted. Examples of heterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl (azetadinyl), tetrahydrofuryl, tetrahydropyranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxatrolyl, diazepinyl, tropanyl, oxazolidinonyl, and homoscopanyl (homotropanyl).

The term "hydroxyalkyl" means an alkyl group as defined above wherein the alkyl group is substituted with one or more OH groups. Examples of hydroxyalkyl groups include HO-CH₂-、HO-CH₂-CH₂-and CH₃-CH(OH)-。

The term "haloalkyl" as used herein refers to an alkyl group, as defined herein, which is substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl and the like.

The term "haloalkoxy" as used herein refers to an alkoxy group, as defined herein, which is substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, trichloromethoxy, and the like.

The term "cyano" as used herein means a substituent having a carbon atom attached to a nitrogen atom through a triple bond, e.g., C ≡ N.

"spirocycloalkyl" or "spirocyclic" means a carbon-generating carbocyclic ring system having two rings connected by a single atom. The size and nature of the rings may be different or the size and nature of the rings may be the same. Examples include spiropentane, spirohexane, spiroheptane, spirooctane, spirononane or spirodecane. One or both of the ringsMay be fused to another cyclic carbocyclic, heterocyclic, aromatic or heteroaromatic ring. One or more of the carbon atoms in the spiro ring may be substituted with a heteroatom (e.g., O, N, S or P). (C)₃-C₁₂) Spirocycloalkyl is a spiro ring containing between 3 and 12 carbon atoms. One or more of these carbon atoms may be substituted with a heteroatom.

The term "spiroheterocycloalkyl" or "spiroheterocyclyl" is understood to mean a spirocyclic ring in which at least one ring is heterocyclic (e.g., at least one ring is furyl, morpholinyl, or piperidinyl).

The term "solvate" refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents for the purposes of this disclosure may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, MeOH, EtOH, and AcOH. Solvates in which water is the solvent molecule are commonly referred to as hydrates. Hydrates include compositions containing a stoichiometric amount of water, as well as compositions containing variable amounts of water.

The term "isomers" refers to compounds having the same composition and molecular weight but differing in physical and/or chemical properties. The structural difference may be in the ability to construct (geometric isomers) or rotate the plane of polarized light (stereoisomers). With respect to stereoisomers, the compounds of formula (I) may have one or more asymmetric carbon atoms and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers.

The disclosure also includes pharmaceutical compositions comprising an effective amount of the disclosed compounds and a pharmaceutically acceptable carrier. Representative "pharmaceutically acceptable salts" include, for example, water soluble and water insoluble salts such as acetate, astragaloside (4, 4-diaminostilbene-2, 2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorsulfonate (camsylate), carbonate, chloride, citrate, clavulanate, dihydrochloride, edetate, edisylate, propionate laurylsulfate, ethanesulfonate, fumarate, fianarate, glucoheptonate, gluconate, glutamate, glycollylarsanilate (glycopyrrolylate), hexafluorophosphate, hexylresorcinate, hydrabamine (hydrabamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, dihydrocarb-mate, and the like, Lactobionate, laurate, magnesium, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methylsulfate, mucate, naphthalenesulfonate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1, 1-methylene-bis-2-hydroxy-3-naphthoate, embonate (einborate)), pantothenate, phosphate/hydrogen phosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suraminate, tannate, tartrate, theachlorate, tosylate, triethyliodide and valerate.

A "patient" or "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate (e.g., monkey, chimpanzee, baboon, or rhesus monkey).

When used in combination with a compound, an "effective amount" is an amount effective to treat or prevent a disease in a subject as described herein.

The term "carrier" as used in this disclosure encompasses carriers, excipients, and diluents, and means a material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ or portion of a subject's body to another organ or portion of the body.

With respect to a subject, the term "treating" refers to ameliorating at least one symptom of a disorder in the subject. Treatment includes curing, ameliorating, or at least partially alleviating the disorder.

The term "disorder" as used in the present disclosure means, and is used interchangeably with, a disease, condition, or illness unless otherwise indicated.

The term "administering" or "administration" as used in this disclosure refers to either directly administering a disclosed compound, or a pharmaceutically acceptable salt of a disclosed compound, or a composition, to a subject, or administering a prodrug derivative or analog of a compound, or a pharmaceutically acceptable salt of a compound, or a composition, to a subject, which can form an equivalent amount of the active compound in the subject.

The term "prodrug" as used in the present disclosure means a compound that is convertible in vivo by metabolic means (e.g., by hydrolysis) to the disclosed compound.

The term "cancer" includes, but is not limited to, the following cancers: bladder cancer, breast cancer (e.g., ductal carcinoma), cervical cancer (e.g., squamous cell carcinoma), colorectal cancer (e.g., adenocarcinoma), esophageal cancer (e.g., squamous cell carcinoma), gastric cancer (e.g., adenocarcinoma, medulloblastoma, colon cancer, choriocarcinoma, squamous cell carcinoma), head and neck cancer, hematological cancer (e.g., acute lymphocytic anemia, acute myelogenous leukemia, acute lymphocytic B-cell leukemia, anaplastic large cell lymphoma, B-cell lymphoma, Burkitt's lymphoma), chronic lymphocytic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic myelogenous leukemia, Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia), lung cancer (e.g., bronchioloalveolar adenocarcinoma, Mesothelioma, mucoepidermoid carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma, squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), lymphoma, nervous system cancer (e.g., glioblastoma, neuroblastoma, glioma), ovarian cancer (e.g., adenocarcinoma), pancreatic cancer (e.g., ductal carcinoma), prostate cancer (e.g., adenocarcinoma), kidney cancer (e.g., renal cell carcinoma, clear cell renal carcinoma), sarcoma (e.g., chondrosarcoma, ewing sarcoma, fibrosarcoma, pluripotent sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (e.g., melanoma, epidermoid carcinoma, squamous cell carcinoma), thyroid cancer (e.g., medullary carcinoma), and uterine cancer.

As used herein, the term "autoimmune disease" or "autoimmune disorder" refers to a condition that is immune-mediated as a result of an attack on self tissue (e.g., when a subject's own antibodies react with host tissue), but may also involve an immune response to a microorganism. Examples of autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, crohn's disease, rheumatoid arthritis and polyarthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosus, cutaneous lupus erythematosus including chilblain lupus erythematosus, lupus nephritis, discoid lupus, subacute cutaneous lupus erythematosus, dermatomyositis, polymyositis, idiopathic myxedema, hashimoto's disease, guillain-barre syndrome, graves 'disease, myasthenia gravis, Sjogren's syndrome, nodular arteritis, autoimmune enteropathy, uveitis, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, proliferative nephropathies, viskott-Aldrich syndrome (Wiskott-Aldrich syndrome), Autoimmune lymphoproliferative syndrome, chronic arthritis, inflammatory chronic sinusitis, colitis, celiac disease, inflammatory bowel disease, Barrett's esophagitis, inflammatory gastritis, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune encephalitis, and autoimmune-mediated hematologic disorders.

The present disclosure relates to compounds capable of inhibiting USP28 and/or USP25, or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers or tautomers thereof, which are useful for treating diseases and disorders associated with the modulation of USP28 and/or USP25 enzymes. The present disclosure further relates to compounds useful for inhibiting USP28 and/or USP25, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

In any embodiment of the invention, the cancer may be any cancer in any organ, for example, the cancer is selected from the group consisting of: glioma, thyroid cancer, breast cancer, small cell lung cancer, non-small cell cancer, gastric cancer, colon cancer, gastrointestinal stromal cancer, pancreatic cancer, bile duct cancer, CNS cancer, ovarian cancer, endometrial cancer, prostate cancer, renal cancer, anaplastic large cell lymphoma, leukemia, multiple myeloma, mesothelioma, and melanoma, and combinations thereof.

The present invention relates to compounds capable of inhibiting USP28 and/or USP25, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof, useful for the treatment of diseases and disorders associated with the modulation of the USP28 and/or USP25 enzymes. The present invention further relates to compounds useful for inhibiting USP28 and/or USP25, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

In one embodiment, the compound of formula (I) has the structure of formula (Ia)

And pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ib):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ic):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Id):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ie):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (If):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ig):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ih):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ii):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In another embodiment, the compound of formula (I) has the structure of formula (Ij):

and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and tautomers thereof.

In some embodiments of the above formula, X is CR₆. In another embodiment, X is N.

In some embodiments of the above formula, R₁Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₉R₁₀. In another embodiment, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, -CN, or-NR₈R₉. In yet anotherIn embodiments, R₁Is H, (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, (C)₁-C₃) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, -CN or-NR₈R₉. In yet another embodiment, R₁Is H, (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, (C)₁-C₃) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₈R₉. In yet another embodiment, R₁Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, -CN, or-NR₈R₉. In yet another embodiment, R₁Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₈R₉. In yet another embodiment, R₁Is H or (C)₁-C₃) An alkyl group. In another embodiment, R₁Is (C)₁-C₃) An alkyl group. In yet another embodiment, R₁Is H, methyl, ethyl, n-propyl, or isopropyl. In another embodiment, R₁Is H, methyl, or ethyl. In yet another embodiment, R₁Is methyl or ethyl.

In some embodiments of the above formula, R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-R₁₀R₁₁. In another embodiment, R₂Is H, (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, (C)₁-C₃) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-R₁₀R₁₁. In yet another embodiment, R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₁₀R₁₁. In another embodiment, R₂Is H, (C)₁-C₃) Alkyl, (C)₁-C₃) Haloalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₁₀R₁₁. In yet another embodiment, R₂Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-R₁₀R₁₁. In another embodiment, R₂Is H or halogen. In another embodiment, R₂Is H.

In another embodiment, R₁And R₂Together form an optionally substituted one to three R₁₂Substituted (C)₄-C₈) A cycloalkyl group. In yet another embodiment, R₁And R₂Together form an optionally substituted one to three R₁₂Substituted (C)₄-C₆) A cycloalkyl group.

In some embodiments of the above formula, R₁Or R₂Is not H.

In some embodiments of the above formula, R₁Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₈R₉(ii) a And R is₂Is H, (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, (C)₁-C₃) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-R₁₀R₁₁。

In some embodiments of the above formula, R₃Is H, (C)₁-C₃) Alkyl, or

(C₁-C₃) A haloalkyl group. In another embodiment, R₃Is H or (C)₁-C₃) An alkyl group. In yet another embodiment, R₃Is H. In another embodiment, R₃Is H, methyl, ethyl, n-propyl, or isopropyl. In yet another embodiment, R₃Is H or methyl.

In some embodiments of the above formula, R₄Is H, (C)₁-C₃) Alkyl, halogen, or

(C₁-C₃) A haloalkyl group. In another embodiment, R₄Is H, halogen, or (C)₁-C₃) An alkyl group. In yet another embodiment, R₄Is H or (C)₁-C₃) An alkyl group. In yet another embodiment, R₄Is H. In another embodiment, R₄Is H, methyl, ethyl, n-propyl, or isopropyl. In yet another embodiment, R₄Is H, F, Cl, methyl, ethyl, n-propyl, or isopropyl. In another embodiment, R₄H, F, or methyl. In yet another embodiment, R₄Is H or methyl.

In some embodiments of the above formula, R_4′Is H, (C)₁-C₃) Alkyl, halogen, or (C)₁-C₃) A haloalkyl group. In another embodiment, R_4′Is H, halogen, or (C)₁-C₃) An alkyl group. In yet another embodiment, R_4′Is H or (C)₁-C₃) An alkyl group. In yet another embodiment, R_4′Is H. In another embodiment, R_4′Is H, methyl, ethyl, n-propyl, or isopropyl. In yet another embodiment, R_4′Is H, fluoro, chloro, methyl, ethyl, n-propyl, or isopropyl. In another embodiment, R_4′H, F, or methyl. In yet another embodiment, R_4′Is H or methyl.

In some embodiments of the above formula, R₅Is- (C)₀-C₃) alkylene-C (O) OH, - (C)₀-C₃) Alkylene-heterocycloalkyl, - (C)₀-C₃) Alkylene-aryl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl wherein heterocycloalkyl, aryl and heteroaryl are optionally substituted with one to three R₁₃And (4) substitution. In another embodiment, R₅Is- (C)₀-C₃) alkylene-C (O) OH, - (C)₀-C₃) Alkylene-heterocycloalkyl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl wherein heterocycloalkyl and heteroaryl are optionally substituted with one to three R₁₃And (4) substitution. In another embodiment, R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl wherein heterocycloalkyl and heteroaryl are optionally substituted with one to three R₁₃And (4) substitution. In another embodiment, R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, in which heterocycloalkyl and heteroaryl are optionally substituted by one toThree R₁₃And (4) substitution. In another embodiment, R₅Is optionally substituted by one to three R₁₃substituted-O-heterocycloalkyl.

In some embodiments of the above formula, R₆Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, halogen, -OH, -CN, (C)₃-C₈) Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein alkyl is optionally substituted with one or more (C)₁-C₄) Alkoxy or-OH, and wherein cycloalkyl, heterocycloalkyl, aryl, and heteroaryl are optionally substituted with one to three R₁₄And (4) substitution. In another embodiment, R₆Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, halogen, -OH, -CN, (C)₃-C₈) Cycloalkyl, aryl, or heteroaryl, wherein alkyl is optionally substituted with one or more (C)₁-C₄) Alkoxy or-OH, and wherein cycloalkyl, aryl, and heteroaryl are optionally substituted with one to three R₁₄And (4) substitution.

In another embodiment, R₅And R₆When on adjacent atoms together form an optionally substituted one to three R₁₅Substituted (C)₃-C₈) A cycloalkyl ring. In yet another embodiment, R₅And R₆When on adjacent atoms together form an optionally substituted one to three R₁₅A substituted heterocycloalkyl ring. In another embodiment, R₅And R₆When on adjacent atoms together form an optionally substituted one to three R₁₅A substituted aryl ring. In yet another embodiment, R₅And R₆When on adjacent atoms together form an optionally substituted one to three R₁₅A substituted heteroaryl ring.

In another embodiment, two R are₆When on adjacent atoms form together (C)₃-C₈) A cycloalkyl ring. In yet another embodiment, two R are₆When on adjacent atoms, together form a heterocycloalkyl ring. In another embodiment, two R are₆When on adjacent atoms together form an aryl ring. In yet another embodiment, two R are₆When on adjacent atoms, together form a heteroaryl ring.

In some embodiments of the above formula, R₇Is H or (C)₁-C₃) An alkyl group. In another embodiment, R₇Is H. In yet another embodiment, R₇Is (C)₁-C₃) An alkyl group. In another embodiment, R₇Is H, methyl, ethyl, n-propyl, or isopropyl. In another embodiment, R₇Is H, methyl, or ethyl.

In some embodiments of the above formula, R₈Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₃) An alkyl group. In another embodiment, R₈Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₂) An alkyl group. In yet another embodiment, R₈Is H, methyl, ethyl, n-propyl, isopropyl, -C (O) CH₃or-C (O) CH₂CH₃。

In some embodiments of the above formula, R₉Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₃) An alkyl group. In another embodiment, R₉Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₂) An alkyl group. In yet another embodiment, R₉Is H, methyl, ethyl, n-propyl, isopropyl, -C (O) CH₃or-C (O) CH₂CH₃。

In some embodiments of the above formula, R₁₀Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₃) An alkyl group. In another embodiment, R₁₀Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₂) An alkyl group. In yet another embodimentIn the embodiment, R₁₀Is H, methyl, ethyl, n-propyl, isopropyl, -C (O) CH₃or-C (O) CH₂CH₃. In another embodiment, R₁₀Is H.

In some embodiments of the above formula, R₁₁Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₃) An alkyl group. In another embodiment, R₁₁Is H, (C)₁-C₃) Alkyl, or-C (O) (C)₁-C₂) An alkyl group. In yet another embodiment, R₁₁Is H, methyl, ethyl, n-propyl, isopropyl, -C (O) CH₃or-C (O) CH₂CH₃. In another embodiment, R₁₁Is H.

In some embodiments of the above formula, R₁₂Is (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, halogen, or-OH. In another embodiment, R₁₂Is (C)₁-C₃) Alkyl, (C)₁-C₃) Haloalkyl, halogen, or-OH. In yet another embodiment, R₁₂Is (C)₁-C₃) Alkyl, halogen, or-OH. In another embodiment, R₁₂Is (C)₁-C₃) Alkyl or-OH. In yet another embodiment, R₁₂is-OH.

In some embodiments of the above formula, R₁₃Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, - (O) NR₁₈R₁₉、-S(O)₂(C₁-C₆) Alkyl, -OH, or-NR₁₆R₁₇Wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₆) Alkoxy, OH, and heterocycloalkyl. In addition toIn one embodiment, R₁₃Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -C (O) NR₁₈R₁₉、-S(O)₂(C₁-C₆) Alkyl, -OH, or-NR₁₆R₁₇Wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₆) Alkoxy, OH, and heterocycloalkyl. In yet another embodiment, R₁₃Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₆R₁₇Wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₆) Alkoxy, OH, and heterocycloalkyl. In another embodiment, R₁₃Is (C)₁-C₄) Alkyl, (C)₃-C₈) Cycloalkyl, -C (O) NR₁₈R₁₉、-S(O)₂(C₁-C₆) Alkyl, -OH, or-NR₁₆R₁₇Wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₆) Alkoxy, OH, and heterocycloalkyl. In yet another embodiment, R₁₃Is (C)₁-C₄) Alkyl, (C)₃-C₈) Cycloalkyl, or-NR₁₆R₁₇Wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₆) Alkoxy, OH, and heterocycloalkyl.

In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together may form-C ═ (O) when attached to the same carbon. In yet another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkylor-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one to three R₂₀Substituted (C)₃-C₈) Spirocycloalkyl. In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one to three R₂₀Substituted (C)₃-C₈) Spiroheterocycloalkyl. In another embodiment, two R are₁₃When on adjacent atoms together form an optionally substituted one to three R₂₀A substituted heterocycloalkyl ring. In another embodiment, two R are₁₃When on adjacent atoms together form an optionally substituted one to three R₂₀A substituted heteroaryl ring. In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together with the atoms to which they are attached may form optionally substituted by one to three R₂₀A substituted bridged heterocycloalkyl ring.

In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together may form-C ═ (O) when attached to the same carbon. In yet another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spirocycloalkyl. In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene oxideWhen alkyl-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spiroheterocycloalkyl. In another embodiment, when R₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl, -O-heterocycloalkyl, or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together with the atoms to which they are attached may form optionally substituted by one or more R₂₀A substituted bridged heterocycloalkyl ring.

In some embodiments of the above formula, R₁₄Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, halogen, cycloalkyl, heterocycloalkyl, or-c (o) -heterocycloalkyl, wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₄) Alkoxy and-OH. In another embodiment, R₁₄Is (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, heterocycloalkyl, or-c (o) -heterocycloalkyl, wherein alkyl is optionally substituted with one to three substituents independently selected from: (C)₁-C₄) Alkoxy and-OH.

In some embodiments of the above formula, R₁₅Is (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, halogen, -OH, -CN, -C (O) OH, or-C (O) O (C)₁-C₄) An alkyl group. In another embodiment, R₁₅Is (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, -OH, -CN, -C (O) OH, or-C (O) O (C)₁-C₄) An alkyl group. In yet another embodiment, R₁₅Is (C)₁-C₄) Alkyl, halogen, -OH, -CN, -C (O) OH, or-C (O) O (C)₁-C₄) An alkyl group. In another embodiment, R₁₅Is (C)₁-C₄) Alkyl, -OH, -C (O) OH, or-C (O) O (C)₁-C₄) An alkyl group. In another embodiment, R₁₅is-C (O) OH, or-C (O) O (C)₁-C₄) An alkyl group.

In some embodiments of the above formula, R₁₆Is H, (C)₁-C₄) Alkyl, (C)₃-C₈) Cycloalkyl, -CH₂C(O)NH₂、-S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl or-C (O) (C)₁-C₄) An alkyl group. In another embodiment, R₁₆Is H, (C)₁-C₄) Alkyl, (C)₃-C₆) Cycloalkyl, -CH₂C(O)NH₂、 -S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl, or-C (O) (C)₁-C₄) An alkyl group. In yet another embodiment, R₁₆Is H, (C)₁-C₄) Alkyl or (C)₃-C₆) A cycloalkyl group. In another embodiment, R₁₆Is H, (C)₁-C₄) alkyl-CH₂C(O)NH₂、-S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl, or-C (O) (C)₁-C₄) An alkyl group.

In some embodiments of the above formula, R₁₇Is H, (C)₁-C₄) Alkyl, (C)₃-C₈) Cycloalkyl, -CH₂C(O)NH₂、-S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl or-C (O) (C)₁-C₄) An alkyl group. In another embodiment, R₁₇Is H, (C)₁-C₄) Alkyl, (C)₃-C₆) Cycloalkyl, -CH₂C(O)NH₂、 -S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl, or-C (O) (C)₁-C₄) An alkyl group. In yet another embodiment, R₁₇Is H, (C)₁-C₄) Alkyl or (C)₃-C₆) A cycloalkyl group. In another embodiment, R₁₇Is H, (C)₁-C₄) Alkyl, -CH₂C(O)NH₂、-S(O)₂(C₁-C₄) Alkyl, -S (O)₂(C₆-C₁₀) Aryl, or-C (O) (C)₁-C₄) An alkyl group.

In some embodiments of the above formula, R₁₈Is H or (C)₁-C₃) An alkyl group. In another embodiment, R₁₈Is H, methyl, ethyl, n-propyl, or isopropyl.

In some embodiments of the above formula, R₁₉Is H or (C)₁-C₃) An alkyl group. In another embodiment, R₁₉Is H, methyl, ethyl, n-propyl, or isopropyl.

In some embodiments of the above formula, R₂₀Is (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, or halogen. In another embodiment, R₂₀Is (C)₁-C₂) Alkyl, (C)₁-C₂) Alkoxy group, (C)₁-C₂) Haloalkyl, (C)₁-C₂) Haloalkoxy, or halogen. In yet another embodiment, R₂₀Is (C)₁-C₂) Alkyl, (C)₁-C₂) Haloalkyl, or halogen. In another embodiment, R₂₀Is (C)₁-C₂) Alkyl, or (C)₁-C₂) A haloalkyl group. In another embodiment, R₂₀Is (C)₁-C₃) An alkyl group. In another embodiment, R₂₀Is methyl, ethyl, n-propyl, or isopropyl.

In another embodiment, two R are₂₀Together when attached to the same carbon form-C ═ (O).

In some embodiments of the above formula, n is 0, 1, or 2. In another embodiment, n is 0 or 1. In yet another embodiment, n is 1, 2, or 3. In another embodiment, n is 1 or 2. In another embodiment, n is 2 or 3. In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3.

In some embodiments of the above formula, R₅Is- (C)₀-C₃) alkylene-C (O) OH, - (C)₀-C₃) Alkylene-heterocycloalkyl, - (C)₀-C₃) Alkylene-aryl, - (C)₀-C₃) Alkylene-heteroaryl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, wherein heterocycloalkyl, aryl and heteroaryl are optionally substituted by one or more R₁₃Substitution; and is

Each R₁₃Independently at each occurrence is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -C (O) NR₁₈R₁₉、-S(O)₂(C₁-C₆) Alkyl, -OH or-NR₁₆R₁₇Wherein the alkyl is optionally substituted with one or more substituents independently selected from: (C)₁-C₆) Alkoxy, OH and heterocycloalkyl; or

When R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together may form-C ═ (O) when attached to the same carbon; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spiro cycloalkyl; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃When attached to the same atom form together optionally substituted by one or more R₂₀Substituted (C)₃-C₈) Spiroheterocycloalkyl; or two R₁₃When on adjacent atoms together form an optionally substituted one or more R₂₀A substituted heterocycloalkyl ring; or two R₁₃When on adjacent atoms together form an optionally substituted one or more R₂₀A substituted heteroaryl ring; or when R is₅Is- (C)₀-C₃) Alkylene-heterocycloalkyl or-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl, two R₁₃Together with the atoms to which they are attached may form optionally substituted by one or more R₂₀A substituted bridged heterocycloalkyl ring.

In some embodiments of the above formula, X is CH.

In some embodiments of the above formula, R₃Is H or CH₃And R is₄Is H or CH₃。

In some embodiments of the above formula, R₃Is H or CH₃，R₄Is H or CH₃And R is_4′Is H, fluorine, or CH₃。

In some embodiments of the above formula, R₂Is H, (C)₁-C₆) Alkyl, aryl, heteroaryl, and heteroaryl,

(C₁-C₆) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NH₂。

In some embodiments of the above formula, R₁And R₂Together form optionally substituted by one or more R₁₂Substituted (C)₃-C₈) A cycloalkyl group.

In some embodiments of the above formula, R₁Is H and R₂Is (C)₁-C₆) An alkyl group.

In some embodiments of the above formula, R₁Is (C)₁-C₆) Alkyl and R₂Is H。

In some embodiments of the above formula, R₁Or R₂Only one of which is H.

In some embodiments of the above formula, R₁Is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -CN, or-NR₈R₉And R is₂Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁。

In some embodiments of the above formula, R₁Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -CN, or-NR₈R₉And R is₂Is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁。

In some embodiments of the above formula, R₁Or R₂One of which is H and the other is (C)₁-C₆) An alkyl group.

In another embodiment, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl radicalHalogen, (C)₃-C₆) Cycloalkyl, or-NR₈R₉(ii) a And R is₂Is (C)₁-C₃) Alkyl, (C)₁-C₃) Alkoxy group, (C)₁-C₃) Haloalkyl, (C)₁-C₃) Haloalkoxy, (C)₁-C₃) Hydroxyalkyl, halogen, (C)₃-C₆) Cycloalkyl, or-NR₁₀R₁₁。

In some embodiments of the above formula, X is CR₆. In another embodiment, X is CR₆And R is₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉. In yet another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉And R is₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁. In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉， R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁And R is₃Is H or (C)₁-C₃) An alkyl group. In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl, and R₄Is H or (C)₁-C₃) An alkyl group. In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl, and R_4′Is H or (C)₁-C₃) An alkyl group.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-heterocycloalkyl.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is- (C)₀-C₃) alkylene-C (O) OH.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-heteroaryl.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃substituted-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-aryl groups.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅Substituted (C)₄-C₈) A cycloalkyl ring.

In another embodiment, X is CR₆，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅A substituted heterocycloalkyl ring.

In some embodiments of the above formula, X is N. In another embodiment, X is N and R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉. In yet another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉And R is₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁. In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁And R is₃Is H or (C)₁-C₃) An alkyl group. In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl, and R₄Is H or (C)₁-C₃) An alkyl group. In yet another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl, and R_4′Is H or (C)₁-C₃) An alkyl group.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-heterocycloalkyl.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) An alkyl group, a carboxyl group,R₄is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is- (C)₀-C₃) alkylene-C (O) OH.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-heteroaryl.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃substituted-N (R)₇)-(C₀-C₃) Alkylene-heterocycloalkyl.

In another embodiment, X is N，R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅Is optionally substituted by one or more R₁₃Substituted- (C)₀-C₃) Alkylene-aryl groups.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅Substituted (C)₄-C₈) A cycloalkyl ring.

In another embodiment, X is N, R₁Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy group, (C)₁-C₄) Haloalkyl, (C)₁-C₄) Haloalkoxy, (C)₁-C₄) Hydroxyalkyl, haloElement, (C)₃-C₈) Cycloalkyl, or-NR₈R₉，R₂Is H, (C)₁-C₄) Alkyl, (C)₁-C₄) Haloalkyl, halogen, (C)₃-C₈) Cycloalkyl, or-NR₁₀R₁₁，R₃Is H or (C)₁-C₃) Alkyl radical, R₄Is H or (C)₁-C₃) Alkyl radical, R_4′Is H or (C)₁-C₃) Alkyl, and R₅And R₆When on adjacent atoms together form an optionally substituted one or more R₁₅A substituted heterocycloalkyl ring.

Non-limiting illustrative compounds of the present disclosure include:

and

in another embodiment of the disclosure, the compound of formula (I) is an enantiomer. In some embodiments, the compound is the (S) -enantiomer. In other embodiments, the compound is the (R) -enantiomer. In yet other embodiments, the compound of formula (I) may be the (+) or (-) enantiomer.

It is to be understood that all isomeric forms are included in the present disclosure, including mixtures thereof. If the compound contains a double bond, the substituent may be in the E or Z configuration. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may have either the cis or trans configuration. All tautomeric forms are also intended to be included.

The compounds of the present disclosure and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, and prodrugs thereof can exist in their tautomeric forms (e.g., as amides or imino ethers). All such tautomeric forms are contemplated herein as part of the present disclosure.

The compounds of the present disclosure may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present disclosure, as well as mixtures thereof (including racemic mixtures), form a part of the present disclosure. In addition, the present disclosure includes all geometric and positional isomers. For example, if a compound of the present disclosure contains a double bond or fused ring, both the cis and trans forms, as well as mixtures, are included within the scope of the present disclosure. Each compound disclosed herein includes all enantiomers that conform to the general structure of the compound. The compounds may be in racemic or enantiomerically pure form, or in any other form with respect to stereochemistry. The results of the assay may be reflected in data collected in racemic form, enantiomerically pure form, or in any other form in terms of stereochemistry.

Mixtures of diastereomers may be separated into their respective diastereomers on the basis of their physicochemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by: the enantiomeric mixtures are converted into diastereomeric mixtures by reaction with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), the diastereomers are separated and the respective diastereomers are converted (e.g., hydrolyzed) into the corresponding pure enantiomers. In addition, some compounds of the present disclosure may be atropisomers (e.g., substituted biaryls) and are considered part of the present disclosure. Enantiomers can also be separated by using a chiral HPLC column.

It is also possible that the compounds of the disclosure may exist in different tautomeric forms, and all such forms are included within the scope of the disclosure. Further, for example, all keto-enol and imine-enamine forms of the compounds are included in the disclosure.

All stereoisomers (e.g., geometric isomers, optical isomers, etc.) (including those of salts, solvates, esters, and prodrugs of the compounds, and salts, solvates, and esters of prodrugs) of the compounds of the present invention, such as those that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may even exist in the absence of an asymmetric carbon), rotameric forms, atropisomers, and diastereomeric forms, are also included within the scope of the disclosure, as are positional isomers (such as, for example, 4-pyridyl and 3-pyridyl). Also, for example, all keto-enol and imine-enamine forms of the compounds are included in the disclosure.) individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers, or may be mixed, for example, as racemates or with all other stereoisomers or selected other stereoisomers. The chiral centers of the present disclosure may have the S or R configuration as defined by IUPAC 1974 recommends. The use of the terms "salt", "solvate", "ester", "prodrug" and the like is intended to apply equally to the salts, solvates, esters and prodrugs of the enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the compounds of the present invention.

The compounds of formula I may form salts, which are also within the scope of the present disclosure. Unless otherwise indicated, reference to a compound of the formula described herein is understood to include reference to a salt thereof.

The present disclosure relates to compounds that are USP28 and/or USP25 modulators. In one embodiment, the compound of the present disclosure is an inhibitor of USP28 and/or USP 25.

The present disclosure relates to compounds as described herein and pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, or tautomers thereof, as well as pharmaceutical compositions comprising one or more compounds as described herein or pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers, or tautomers thereof.

Method for synthesizing compound

The compounds of the present disclosure can be prepared by a variety of methods, including standard chemistry. Suitable synthetic routes are depicted in the schemes given below.

The compounds of formula (I) may be prepared by methods known in the art of organic synthesis, as illustrated by the synthetic schemes below. In the schemes described below, it is readily understood that protective groups for sensitive or reactive groups are used as necessary according to general principles or chemistry. The protecting Groups were manipulated according to standard methods of Organic Synthesis (T.W.Greene and P.G.M.Wuts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999). These groups are removed at a convenient stage of the compound synthesis, as will be apparent to those skilled in the art. The procedure chosen, as well as the reaction conditions and the order in which they are carried out, should be in accordance with the preparation of the compounds of formula (I).

One skilled in the art will recognize whether a stereocenter is present in the compound of formula (I). Thus, the present disclosure includes both possible stereoisomers (unless specified in the synthesis) and includes not only the racemic compound, but also the respective enantiomers and/or diastereomers. When a compound is desired as a single enantiomer or diastereomer, it may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. The resolution of the final product, intermediate or starting material may be effected by any suitable means known in the art. See, e.g., "Stereochemistry of Organic Compounds" by E.L.Eliel, S.H. Wilen, and L.N.Mander (Wiley-lnterscience, 1994).

The compounds described herein can be made from commercially available starting materials or synthesized using known organic, inorganic, and/or enzymatic processes.

Preparation of the Compounds

The compounds of the present invention can be prepared in various ways well known to those skilled in the art of organic synthesis. By way of example, the compounds of the present invention may be synthesized using the methods described below, as well as synthetic methods known in the art of synthetic organic chemistry, or variations thereof as understood by those skilled in the art. Preferred methods include, but are not limited to, those described below. The compounds of the invention may be synthesized according to the procedures outlined in general scheme 1, including assembly intermediates 2a and 2 b. The starting materials are either commercially available or prepared by known procedures in the reported literature or as shown below.

General scheme 1

Wherein R is₁-R₄、R_4′、R₅、R₆And n is as defined for formula (I).

The general manner of preparing the target compounds of formula (I) by using intermediates 2a and 2b is outlined in general scheme 1 above. Coupling agents (e.g., 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide with 1-hydroxybenzotriazole (EDCI/HOBt), (benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP), O-benzotriazole-N, N, N' -tetramethyl-uronium-Hexafluorophosphate (HBTU) or [ bis (dimethylamino) methylene ] -1H-1, 2, 3-triazolo [4, 5-b ] pyridinium-3-oxide Hexafluorophosphate (HATU), and a base (e.g., Triethylamine (TEA), N, N-Diisopropylethylamine (DIEA) or 4-Dimethylaminopyridine (DMAP)) are used in a solvent (e.g., DCM, DMF, etc.), coupling of carboxylic acid 2a with amine 2b under standard amide forming conditions provides the desired product of formula (I).

The compounds of formula (I) may exist as enantiomers or diastereoisomers. Enantiomerically pure compounds of formula (I) can be prepared using enantiomerically pure chiral building blocks. Alternatively, a racemic mixture of the final compound or a racemic mixture of higher intermediates may be subjected to chiral purification as described herein below to deliver the desired enantiomerically pure intermediate or final compound. In the case of purification of the higher intermediate into its respective enantiomers, each respective enantiomer may be carried separately to deliver the final enantiomerically pure compound of formula (I).

It is to be understood that, unless otherwise indicated, in the description and formulae shown above, various groups R₁-R₄、R_4′、R₅、R₆N and other variables are as defined above. Furthermore, for synthetic purposes, the compounds of general scheme 1 are merely representative of the selection groups to illustrate the general synthetic methods of compounds of formula (I) as defined herein.

Methods of using the disclosed compounds

Another aspect of the disclosure relates to methods of treating, preventing, inhibiting or eliminating diseases or disorders associated with the modulation of USP 28. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP28 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer.

In another aspect, the disclosure relates to methods of treating, preventing, inhibiting, or eliminating a disease or disorder associated with inhibition of USP 28. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP28 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer.

In another aspect, the disclosure relates to a method of inhibiting USP 28. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I).

Another aspect of the disclosure relates to methods of treating, preventing, inhibiting or eliminating diseases or disorders associated with the modulation of USP 25. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP25 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the disclosure relates to methods of treating, preventing, inhibiting, or eliminating a disease or disorder associated with inhibition of USP 28. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP25 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the disclosure relates to a method of inhibiting USP 25. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I).

Another aspect of the disclosure relates to methods of treating, preventing, inhibiting or eliminating diseases or disorders associated with the modulation of USP 25. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP28 and USP25 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the disclosure relates to methods of treating, preventing, inhibiting, or eliminating a disease or disorder associated with inhibition of USP 28. The method comprises administering to a patient in need of treatment of a disease or disorder associated with modulation of USP28 and USP25 an effective amount of a composition and a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the disclosure relates to methods of inhibiting USP28 and USP 25. The method involves administering to a patient in need thereof an effective amount of a compound of formula (I).

Another aspect of the present disclosure relates to a method of treating, preventing, inhibiting or eliminating a disease or disorder in a patient associated with inhibition of USP28, comprising administering to a patient in need thereof an effective amount of a compound of formula (I). In one embodiment, the disease or disorder is cancer.

Another aspect of the present disclosure relates to a method of treating, preventing, inhibiting or eliminating a disease or disorder in a patient associated with inhibition of USP25, comprising administering to a patient in need thereof an effective amount of a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

Another aspect of the present disclosure relates to a method of treating, preventing, inhibiting or eliminating a disease or disorder in a patient associated with inhibition of USP28 and USP25, the method comprising administering to a patient in need thereof an effective amount of a compound of formula (I). In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the invention relates to a method of treating, preventing, inhibiting or eliminating cancer. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the invention relates to a method of treating, preventing, inhibiting or eliminating inflammation. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the invention relates to a method of treating, preventing, inhibiting or eliminating an autoimmune disease. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the invention relates to methods of treating, preventing, inhibiting, or eliminating an infectious disease. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In one embodiment, the infectious disease is a viral infection. In another embodiment, the infectious disease is a bacterial infection.

In another aspect, the invention relates to a method of treating, preventing, inhibiting or eliminating a viral infection. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the invention relates to a method of treating, preventing, inhibiting or eliminating a bacterial infection. The method comprises administering to a patient in need of treatment for cancer an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof, for use in a method of treatment, prevention, inhibition or elimination of a disease or disorder associated with the inhibition of USP 28. In one embodiment, the disease or disorder is cancer.

In another aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treatment, prevention, inhibition, or elimination of a disease or disorder associated with the inhibition of USP 25. In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

Another aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treatment, prevention, inhibition, or elimination of a disease or disorder associated with the inhibition of USP28 and USP 25. In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating cancer.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating inflammation.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating an autoimmune disease.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating an infectious disease.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating a viral infection.

In another aspect, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in a method of treating, preventing, inhibiting, or eliminating a bacterial infection.

Another aspect of the present invention is directed to the use of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of a disease or disorder associated with the inhibition of USP 28. In one embodiment, the disease or disorder is cancer.

Another aspect of the present invention is directed to the use of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of a disease or disorder associated with the inhibition of USP 25. In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

Another aspect of the present invention is directed to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of a disease or disorder associated with the inhibition of USP28 and USP 25. In one embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is inflammation. In another embodiment, the disease or disorder is an autoimmune disease. In another embodiment, the disease or disorder is an infectious disease. In another embodiment, the disease or disorder is a viral infection. In another embodiment, the disease or disorder is a bacterial infection.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of cancer.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of inflammation.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of an autoimmune disorder.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of an infectious disease. In one embodiment, the infectious disease is a viral infection. In another embodiment, the infectious disease is a bacterial infection.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of a viral infection.

In another aspect, the present invention relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in the manufacture of a medicament for the treatment, prevention, inhibition, or elimination of a bacterial infection.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with cancer.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with inflammation.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with autoimmune disease.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with an infectious disease. In one embodiment, the infectious disease is a viral infection. In another embodiment, the infectious disease is a bacterial infection.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with a viral infection.

In other embodiments, the present invention relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or disorder associated with a bacterial infection.

The present disclosure also relates to the use of an inhibitor of USP28 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or condition mediated by USP28, wherein the medicament comprises a compound of formula (I).

The present disclosure also relates to the use of an inhibitor of USP25 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or condition mediated by USP25, wherein the medicament comprises a compound of formula (I).

The present disclosure also relates to the use of an inhibitor of USP28 and USP25 for the preparation of a medicament for use in the treatment, prevention, inhibition or elimination of a disease or condition mediated by USP28 and USP25, wherein the medicament comprises a compound of formula (I).

In another aspect, the present disclosure relates to a method for the manufacture of a medicament for treating, preventing, inhibiting or eliminating a disease or condition mediated by USP28, wherein the medicament comprises a compound of formula (I).

Another aspect of the present disclosure relates to a method for the manufacture of a medicament for treating, preventing, inhibiting or eliminating a disease or condition mediated by USP25, wherein the medicament comprises a compound of formula (I).

In another aspect, the present disclosure relates to a method for the manufacture of a medicament for treating, preventing, inhibiting or eliminating a disease or condition mediated by USP28 and USP25, wherein the medicament comprises a compound of formula (I).

In some embodiments of the methods described herein, the cancer is selected from bladder cancer, breast cancer (e.g., ductal cancer), cervical cancer (e.g., squamous cell carcinoma), colorectal cancer (e.g., adenocarcinoma), colon cancer, esophageal cancer (e.g., squamous cell carcinoma), gastric cancer (e.g., adenocarcinoma, choriocarcinoma, squamous cell carcinoma), head and neck cancer, hematological cancer (e.g., acute lymphocytic anemia, acute myelogenous leukemia, acute lymphocytic B-cell leukemia, anaplastic large cell lymphoma, B-cell lymphoma, Burkitt's lymphoma), chronic lymphocytic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic myelogenous leukemia, Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia), Lung cancer (e.g., bronchioloalveolar adenocarcinoma, mesothelioma, mucoepidermoid carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma, squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), lymphoma, nervous system cancer (e.g., glioblastoma, neuroblastoma, glioma), ovarian cancer (e.g., adenocarcinoma), pancreatic cancer (e.g., ductal carcinoma), prostate cancer (e.g., adenocarcinoma), kidney cancer (e.g., renal cell carcinoma, clear cell renal cell carcinoma), sarcoma (e.g., chondrosarcoma, ewing's sarcoma, fibrosarcoma, pluripotent sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (e.g., melanoma, epidermoid carcinoma, squamous cell carcinoma), thyroid cancer (e.g., medullary carcinoma), and uterine cancer. In some embodiments, the cancer is a cancer sensitive to USP28 inhibition. In other embodiments, the cancer is a cancer sensitive to inhibition of USP 25. In other embodiments, the cancer is a cancer that is sensitive to inhibition by USP28 and USP 25.

In any embodiment of the present disclosure, the cancer may be any cancer in any organ, for example, the cancer is selected from the group consisting of: glioma, thyroid cancer, breast cancer, small cell lung cancer, non-small cell cancer, gastric cancer, colon cancer, gastrointestinal stromal cancer, pancreatic cancer, bile duct cancer, CNS cancer, ovarian cancer, endometrial cancer, prostate cancer, renal cancer, anaplastic large cell lymphoma, leukemia, multiple myeloma, mesothelioma, and melanoma, and combinations thereof.

In other embodiments, the cancer is selected from acute myeloid leukemia, gastric cancer, pancreatic cancer, colorectal cancer, glioblastoma, neuroblastoma, small cell lung cancer, non-small cell lung cancer, and squamous cell carcinoma.

In another embodiment, the disclosure relates to a compound of formula (I), or a pharmaceutical composition comprising a compound of the disclosure and a pharmaceutically acceptable carrier, for use in treating cancer, including, but not limited to, bladder cancer, breast cancer (e.g., ductal cancer), cervical cancer (e.g., squamous cell carcinoma), colorectal cancer (e.g., adenocarcinoma), esophageal cancer (e.g., squamous cell carcinoma), gastric cancer (e.g., adenocarcinoma, choriocarcinoma, squamous cell carcinoma), head and neck cancer, hematologic cancer (e.g., acute lymphocytic anemia, acute myelogenous leukemia, acute lymphocytic B-cell leukemia, anaplastic large cell lymphoma, B-cell lymphoma, Burkitt's lymphoma), chronic lymphocytic leukemia, chronic eosinophilic leukemia/hypereosinophilic syndrome, chronic myelogenous leukemia, and leukemia, Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia, lung cancer (e.g., bronchioloalveolar adenocarcinoma, mesothelioma, mucoepidermoid carcinoma, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), lymphoma, nervous system cancer (e.g., glioblastoma, neuroblastoma, glioma), ovarian cancer (e.g., adenocarcinoma), pancreatic cancer (e.g., ductal carcinoma), prostate cancer (e.g., adenocarcinoma), renal cancer (e.g., renal cell carcinoma, clear cell renal cell carcinoma), sarcoma (e.g., chondrosarcoma, ewing's sarcoma, fibrosarcoma, pluripotent sarcoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma), skin cancer (e.g., melanoma, epidermoid carcinoma, squamous cell carcinoma), thyroid cancer (e.g., medullary carcinoma), and uterine cancer. In other embodiments, the cancer is selected from acute myeloid leukemia, gastric cancer, pancreatic cancer, colorectal cancer, glioblastoma, neuroblastoma, small cell lung cancer, non-small cell lung cancer, and squamous cell carcinoma.

In some embodiments, the patient is selected for treatment based on gene amplification and/or elevated tumor expression of USP28, MYC, LSD1, NICD1 and/or reduced expression of FBXW7 relative to tissue-matched expression.

In some embodiments, the patient is selected for treatment based on gene amplification and/or elevated tumor expression of USP28, USP25, MYC, LSD1, NICD1 and/or reduced expression of FBXW7 relative to tissue-matched expression.

In some embodiments, administration of a compound of formula (I), or a pharmaceutical composition comprising a compound of the disclosure and a pharmaceutically acceptable carrier, induces a change in cell cycle, cell viability, apoptosis, or differentiation.

For example, a change in cell cycle or cell viability or differentiation may be indicated by a decrease in tumor levels of MYC, LSD1, NICD1, PIM1, CDK1, POLA2, HEY1, and/or CCND1, and/or an increase in levels of CD86, p21, LGALS4, and/or DLL 1.

In another embodiment, the disclosure relates to a compound of formula (I) or a pharmaceutical composition comprising a compound of the disclosure and a pharmaceutically acceptable carrier for use in treating autoimmune diseases including, but not limited to, multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, crohn's disease, rheumatoid arthritis and polyarthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosus, cutaneous lupus erythematosus including chilblain lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, dermatomyositis, polymyositis, idiopathic myxoedema, hashimoto's disease, guillain-barre syndrome, graves disease, myasthenia gravis, Sjogren's syndrome, nodular arteritis, sarcoidosis, rheumatoid arthritis, Autoimmune enteropathy, uveitis, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, proliferative glomerulonephritis, visstot-Aldrich syndrome, autoimmune lymphoproliferative syndrome, chronic arthritis, inflammatory chronic sinusitis, colitis, celiac disease, inflammatory enteropathy, Barrett's esophagitis, inflammatory gastritis, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune encephalitis, and autoimmune-mediated hematological diseases.

In any embodiment of the disclosure, the autoimmune disease may be, for example, an autoimmune disease selected from: multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis and polyarthritis, local and systemic scleroderma, systemic lupus erythematosus, discoid lupus erythematosus, cutaneous lupus erythematosus including chilblain lupus erythematosus, lupus nephritis, discoid lupus, subacute cutaneous lupus erythematosus, dermatomyositis, polymyositis, idiopathic myxoedema, Hashimoto's disease, Guillain-Barre syndrome, Grave's disease, myasthenia gravis, Sjogren's syndrome, nodular arteritis, autoimmune bowel disease, uveitis, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, proliferative glomerulonephritis, Wiskott-Aldrich syndrome, Autoimmune lymphoproliferative syndrome, chronic arthritis, inflammatory chronic sinusitis, colitis, celiac disease, inflammatory bowel disease, Barrett's esophagitis, inflammatory gastritis, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune encephalitis, and autoimmune-mediated hematologic disorders.

Another aspect of the present disclosure relates to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise an excipient, diluent or surfactant.

In one embodiment, methods of treating diseases or disorders associated with the modulation of USP28, including cancer, are provided, the methods comprising administering a compound of formula (I) to a patient suffering from at least one of said diseases or disorders.

In another embodiment, methods of treating diseases or disorders associated with the modulation of USP25, including cancer, inflammation, autoimmune diseases, viral infections and bacterial infections, comprising administering a compound of formula (I) to a patient suffering from at least one of said diseases or disorders are provided.

In another embodiment, methods of treating diseases or disorders associated with modulation of USP28 and USP25, including cancer, inflammation, autoimmune diseases, viral infections and bacterial infections, comprising administering a compound of formula (I) to a patient suffering from at least one of said diseases or disorders are provided.

One therapeutic use of a compound or composition of the present disclosure that inhibits USP28 is to provide treatment to a patient or subject suffering from cancer.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide treatment to a patient or subject suffering from cancer.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide a treatment to a patient or subject suffering from cancer.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide a treatment to a patient or subject suffering from inflammation.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide a treatment to a patient or subject suffering from inflammation.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide treatment to a patient or subject suffering from an autoimmune disease.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide treatment to a patient or subject having an autoimmune disease.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide treatment to a patient or subject suffering from an infectious disease.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide a treatment to a patient or subject suffering from an infectious disease.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide treatment to a patient or subject suffering from a viral infection.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide a treatment to a patient or subject having a viral infection.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP25 is to provide a treatment to a patient or subject suffering from a bacterial infection.

Another therapeutic use of a compound or composition of the present disclosure that inhibits USP28 and USP25 is to provide a treatment to a patient or subject having a bacterial infection.

The disclosed compounds of the present disclosure can be administered in an effective amount to treat or prevent a disease and/or arrest its development in a subject.

Administration of the disclosed compounds can be accomplished via any mode of administration of the therapeutic agent. These modes include systemic or topical administration, such as oral, nasal, parenteral, transdermal, subcutaneous, vaginal, buccal, rectal or topical modes of administration.

Depending on the intended mode of administration, the disclosed compositions may be in solid, semi-solid, or liquid dosage forms, such as, for example, injections, tablets, suppositories, pills, time-release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, and the like, sometimes in unit doses and in accordance with conventional pharmaceutical practice. Likewise, they can also be administered in intravenous (bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, and all using forms well known to those skilled in the art of pharmacy.

Exemplary pharmaceutical compositions are tablets and gelatin capsules comprising a compound of the disclosure and a pharmaceutically acceptable carrier, such as a) a diluent, for example, purified water, triglyceride oil (such as hydrogenated or partially hydrogenated vegetable oil, or mixtures thereof), corn oil, olive oil, sunflower oil, safflower oil, fish oil (such as EPA or DHA, or esters or triglycerides thereof, or mixtures thereof), omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose, and/or glycine; b) lubricants, for example silica, talc, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and/or polyethylene glycol; also suitable for tablets; c) binders, for example magnesium aluminium silicate, starch paste, gelatin, gum tragacanth, methyl cellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums (such as gum acacia), gum tragacanth or sodium alginate, waxes and/or polyvinylpyrrolidone, if desired; d) disintegrating agents, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; e) absorbents, colorants, flavors, and sweeteners; f) emulsifying or dispersing agents, such as Tween 80, Labrasol, HPMC, DOSS, capryl 909, labrafac, labrafil, peceol, transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin E TGPS, or other acceptable emulsifying agents; and/or g) agents that enhance absorption of the compound, such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG 200.

Liquid, in particular injectable compositions may be prepared, for example, by dissolution, dispersion, or the like. For example, the disclosed compounds are dissolved in or mixed with a pharmaceutically acceptable solvent (such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like), thereby forming an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron, or serum proteins may be used to solubilize the disclosed compounds.

The disclosed compounds may also be formulated as suppositories, which may be prepared from fatty emulsions or suspensions; polyalkylene glycols such as propylene glycol are used as carriers.

The disclosed compounds may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, the membrane of the lipid component is hydrated with an aqueous solution of the drug to form a lipid layer encapsulating the drug, as described in U.S. patent No. 5,262,564, which is hereby incorporated by reference in its entirety.

The disclosed compounds can also be delivered by using monoclonal antibodies as a separate carrier coupled to the disclosed compounds. The disclosed compounds can also be coupled to soluble polymers as targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide polylysine substituted with palmitoyl residues. In addition, the disclosed compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, such as polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphiphilic hydrogel block copolymers. In one embodiment, the disclosed compounds are not covalently bound to a polymer (e.g., a polycarboxylic acid polymer or a polyacrylate).

Parenteral injectable administration is commonly used for subcutaneous, intramuscular or intravenous injection and infusion. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or in solid form suitable for dissolution in liquid prior to injection.

Another aspect of the present disclosure relates to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may further comprise an excipient, diluent or surfactant.

The compositions may be prepared according to conventional mixing, granulating, or coating methods, respectively, and the pharmaceutical compositions of the invention may contain from about 0.1% to about 99%, from about 5% to about 90%, or from about 1% to about 20%, by weight or volume, of the disclosed compounds.

The dosage regimen utilizing the disclosed compounds is selected in accordance with a variety of factors including the type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; renal or hepatic function of the patient; and the specific compounds disclosed for use. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

When used for the indicated effects, the effective dosage amount of the disclosed compounds ranges from about 0.5mg to about 5000mg of the disclosed compounds required to treat the condition. Compositions for in vivo or in vitro use may contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1000, 1250, 2500, 3500, or 5000mg of the disclosed compound, or, alternatively, in the range of one amount to another in a dosage list. In one embodiment, the composition is in the form of a tablet that may be scored.

Examples

The disclosure is further illustrated by the following examples and synthetic schemes, which should not be construed as limiting the disclosure to the scope or spirit of the specific procedures described herein. It should be understood that these examples are provided to illustrate certain embodiments, and are therefore not intended to limit the scope of the disclosure. It is further understood that various other embodiments, modifications, and equivalents, which may occur to those skilled in the art, may have to be resorted to without departing from the spirit of the disclosure and/or the scope of the appended claims.

Analytical methods, materials and apparatus

Unless otherwise indicated, reagents and solvents were used as received from commercial suppliers. Proton Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker or Varian spectrometer at 300 or 400 MHz. The spectra are given in ppm (δ) and the coupling constants J are reported in hertz. Tetramethylsilane (TMS) was used as an internal standard. Mass spectra were collected using a Waters ZQ Single Quad mass spectrometer (ion trap electrospray ionization (ESI)). Purity and low resolution mass spectral data were measured using a Waters Acquity i-class Ultra Performance Liquid Chromatography (UPLC) system with an Acquity photodiode array detector, an Acquity Evaporative Light Scattering Detector (ELSD) and a Waters ZQ mass spectrometer. Data were obtained using Waters masslynx4.1 software and purity was characterized by UV wavelength 220nm, Evaporative Light Scattering Detection (ELSD) and electrospray ionization (ESI). (column: Acquity UPLC BEH C181.7 μm 2.1X is 50 mm; the flow rate is 0.6 mL/min; solvent A (95%/5%/0.1%: 10mM ammonium formate/acetonitrile/formic acid), solvent B (95%/5%/0.09%: acetonitrile/water/formic acid); gradient: 5% -100% B from 0 to 2min, 100% B to 2.2min, and 5% B at 2.21 min. Using standard solvent gradient programs (e.g., as described below), at 254nm or 220nm, using UV detection (Waters 2489UV/998PDA), on a Waters SunAire C18OBD Prep column (R) ((R))

5 μm, 19mm x50 mm), Waters Xbridge BEH C18OBD Prep column (R) (A.sub.D)

5 μm, 19mm X50 mm), Waters SunAire C18OBD Prep column (C)

5 μm, 19mm x150mm), Waters Xbridge BEH Shield RP18OBDPrep column(s) ((R)

5 μm, 19mm x150mm), or Waters XSelect CSH C18OBD Prep column(s)

5 μm, 19mm x150mm) was purified by preparative HPLC. The absolute configuration of the isolated enantiomers of the compounds of the examples described herein was not determined. Thus, in each case, the configuration of the decomposed material is arbitrarily designated as R or S.

Abbreviations used in the following examples and elsewhere herein are:

atm atmosphere

br broad peak

BINAP (2, 2 '-bis (diphenylphosphino) -1, 1' -binaphthyl)

BOP ((1H-benzo [ d ] [1, 2, 3] triazol-1-yl) oxy) tris (dimethylamino) phosphonium hexafluorophosphate (V)

Cbz carboxybenzyl radical

d double peak

DCM dichloromethane

DEA diethylamine

DIEA N, N-diisopropylethylamine

DMA N, N-dimethylacetamide

DMAP 4-dimethylaminopyridine

DMF N, N-dimethylformamide

DMSO dimethyl sulfoxide

dppf 1, 1' -bis (diphenylphosphino) ferrocene

EA Ethyl acetate

EDCI N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride

ESI electrospray ionization

FCC flash column chromatography

h hours

HATU [ bis (dimethylamino) methylene ] -1H-1, 2, 3-triazolo [4, 5-b ] pyridinium 3-oxide hexafluorophosphate

HBTU 3- [ bis (dimethylamino) methylonium ] -3H-benzotriazole-1-oxide hexafluorophosphate salt

HMPA hexamethylphosphoramide

HOBt benzotriazol-1-ol

HPLC high performance liquid chromatography

LCMS liquid chromatography-mass spectrometry

m multiplet

MHz megahertz

min for

uW microwave

NCS N-chlorosuccinimide

NMR nuclear magnetic resonance

PE Petroleum Ether

parts per million ppm

q quartet peak

RT Room temperature

Ruphos 2-dicyclohexylphosphino-2 ', 6' -diisopropoxybiphenyl

s single peak

SPhos 2-dicyclohexylphosphino-2 ', 6' -dimethoxybiphenyl

t triplet peak

tBuBrettphos di-tert-butyl (2 ', 4', 6 '-triisopropyl-3, 6-dimethoxy- [1, 1' -biphenyl ] -2-yl) phosphine

TEA Triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TosMIC 1- (isocyanomethylsulfonyl) -4-methylbenzene

XantPhos 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene

XPhos 2-dicyclohexylphosphino-2 ', 4 ', 6 ' -triisopropylbiphenyl

Example 1: intermediate 1, 7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxylic acid

Step 1.3-hydroxy-5-methylpyrazine-2-carboxamide

2-oxopropanal (277mL, 40% wt, 1.54mol), NaHSO₃A solution of (201g, 1.93mol) and sodium hydroxide (6.4g, 0.16mol) in water (500mL) was stirred at 80 ℃ for 1 h. 2-Aminomalonamide (150g, 1.28mol) was then added and the mixture was stirred at 80 ℃ for a further 2 h. Sodium acetate (263g, 3.21mol) was added, followed by dropwise addition of H at 50-65 deg.C₂O₂(30%; 210 mL). The reaction mixture was cooled to room temperature and stirred for 1 h. The resulting precipitate was collected by filtration and recrystallized from 75% EtOH to provide 3-hydroxy-5-methylpyrazine-2-carboxamide as an orange solid (119g, 61% yield).¹H-NMR(400MHz，DMSO-d₆)：δ8.75(br s，1H)，8.06(br s，1H)，7.83(br s， 1H)，2.37(s，3H)。

Step 2.3-chloro-5-methylpyrazine-2-carbonitrile

To a mixture of 3-hydroxy-5-methylpyrazine-2-carboxamide (155g, 1.01mol) and TEA (205g, 2.03mol) was added POCl dropwise at 0 deg.C₃(500 mL). The reaction mixture was refluxed for a further 4h and then cooled to room temperature. The mixture was concentrated in vacuo and then ethyl acetate (500mL) and saturated aqueous NaHCO were added₃(1000 mL). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (2 × 500 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography eluting with petroleum ether/EtOAc (95: 5) to provide 3-chloro-5-methylpyrazine-2-carbonitrile as a pale yellow solid (55g, 35% yield).¹H NMR(400MHz，CDCl₃)：δ8.51(s，1H)， 2.69(s，3H)。

Step 3.7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxylic acid methyl ester

To a solution of 3-chloro-5-methylpyrazine-2-carbonitrile (19g, 0.12mol) and methyl 2-mercaptoacetate (14 g, 0.13mol) in THF (200mL) at 0 deg.C was added NaH (60% dispersion in mineral oil; 7.5g, 0.19mol) in small portions. The reaction mixture was stirred at room temperature overnight, and then water (200mL) was carefully added to quench the reaction. The resulting mixture was extracted with ethyl acetate (2 × 300 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified via silica gel column chromatography eluting with petroleum ether/EtOAc (90: 10 to 70: 30) to provide 7-amino-3-methylthioeno [2,3-b ] as a pale yellow solid]Pyrazine-6-carboxylic acid methyl ester (20g, 72% yield).¹H NMR(400MHz， DMSO-d₆)：68.68(s，1H)，7.10(br s，2H)，3.83(s，3H)，2.65(s，3H)。

Step 4.7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxylic acid

To 7-amino-3-methylthioeno [2,3-b ] at room temperature]To a solution of pyrazine-6-carboxylic acid methyl ester (50g, 0.22mol) in THF (200mL) and water (200mL) was added NaOH (13g, 0.34 mol). The reaction mixture was stirred at 50 ℃ for 4h, cooled to room temperature, and then washed with ethyl acetate (2 × 200 mL). The aqueous layer was separated and the pH adjusted to 4-5 with aqueous HCl (2M). Passing the obtained precipitate throughSeparated by filtration and dried to provide 7-amino-3-methylthioeno [2,3-b ] as a yellow solid]Pyrazine-6-carboxylic acid (45 g, 96% yield).¹H NMR(400MHz，DMSO-d₆)：δ12.97(br s，1H)，8.66(s，1H)， 6.96(br s，2H)，2.65(s，3H)。

Example 2: intermediate 2, 7-amino-3-ethylthieno [2,3-b ] pyrazine-6-carboxylic acid

Step 1.3-chloro-2-cyanopyrazine 1-oxide

To a 250-mL 3-necked round-bottomed flask, 3-chloropyrazine-2-carbonitrile (10.0g, 71.7 mmol) and concentrated sulfuric acid (70mL) were added at 0 deg.C, followed by addition of K in portions₂S₂O₈(23.3g, 86.3 mmol). The resulting solution was stirred at 25 ℃ for 24h and then extracted with ethyl acetate (3 × 500 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was triturated with petroleum ether (100mL) and the resulting solid was collected by filtration and dried under vacuum to afford 3-chloro-2-cyanopyrazine 1-oxide (3.4g, 31%) as a yellow solid which continued without further purification. LCMS (ESI, m/z): 156[ M + H ]]⁺。

Step 2.7-amino-6- (methoxycarbonyl) thieno [2,3-b ] pyrazine 1-oxide

To a 250-mL round bottom flask purged and maintained with a nitrogen inert atmosphere was added 3-chloro-2-cyanopyrazine 1-oxide (4.40g, 28.3mmol), methyl 2-mercaptoacetate (3.01g, 28.4mmol), and DMF (40mL) at 0 deg.C, followed by the addition of sodium methoxide (6.13g, 114mmol) in portions. The resulting mixture was stirred at 25 ℃ for 13h and then quenched by the addition of water (150 mL). The resulting solution was extracted with ethyl acetate (3 × 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 100 to 10: 1) to provide 7-amino-6- (methoxycarbonyl) thieno [2,3-b ] as a yellow solid]Pyrazine 1-oxide (2.3g, 36%). LCMS (ESI, m)/z)：226[M+H]⁺。

Step 3.7-amino-2-chlorothieno [2,3-b ] pyrazine-6-carboxylic acid methyl ester

Adding 7-amino-6- (methoxycarbonyl) thieno [2,3-b ] to a 12-mL microwave tube]Pyrazine 1-oxide (0.600g, 2.66mmol) and POCl₃(6 mL). The resulting solution was heated under microwave irradiation at 90 ℃ for 1 h. The reaction mixture was cooled to room temperature and then concentrated in vacuo. The crude product was taken up in water (50mL) and the resulting aqueous mixture was extracted with ethyl acetate (3 × 100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 100 to 10: 1) to provide 7-amino-2-chlorothieno [2,3-b ] as a yellow solid]Pyrazine-6-carboxylic acid methyl ester (200mg, 31%). LCMS (ESI, m/z): 244 [ M + H]⁺。

Step 4.7-amino-2-ethylthieno [2,3-b ] pyrazine-6-carboxylic acid methyl ester

To a 25-mL round bottom flask purged and maintained with a nitrogen inert atmosphere was added 7-amino-2-chlorothieno [2,3-b ]]Pyrazine-6-carboxylic acid methyl ester (0.20g, 0.82mmol), Pd (dppf) Cl₂CH₂Cl₂(0.13g, 0.16mmol), and toluene (7 mL). A solution of diethylzinc in toluene (1.5M, 4.11 mL, 6.16mmol) was then added and the resulting solution was stirred at 80 ℃ for 18 h. The reaction was cooled to room temperature and concentrated in vacuo. The crude product was purified via silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 3) to provide 7-amino-2-ethylthieno [2,3-b ] as a yellow solid]Pyrazine-6-carboxylic acid methyl ester (190mg, 98%). LCMS (ESI, m/z): 238[ M + H ]]⁺。

Step 5.7-amino-2-ethylthieno [2,3-b ] pyrazine-6-carboxylic acid

Adding 7-amino-2-ethylthieno [2,3-b ] to a 25-mL round-bottom flask]Pyrazine-6-carboxylic acid methyl ester (0.190g, 0.80mmol), methanol (3mL), water (1mL), and sodium hydroxide (0.064g, 1.60 mmol). The resulting solution was stirred at 25 ℃ for 18h and then concentrated in vacuo to remove most of the organic solvent. The pH of the solution was adjusted to about 7 with aqueous HCl (1M). Filtering the obtained solidCollected and dried in vacuo to provide 7-amino-2-ethylthieno [2,3-b ] as a yellow solid]Pyrazine-6-carboxylic acid (125mg, 70%). LCMS (ESI, m/z): 224[ M + H]⁺。

Example 3: intermediate 3.4- (4- (2-aminoethyl) phenyl) piperazine-1-carboxylic acid benzyl ester (hydrochloride)

Step 1. (4-Bromophenylethyl) carbamic acid tert-butyl ester

To a solution of 2- (4-bromophenyl) ethan-1-amine (5.00g, 25.0mmol) in anhydrous dichloromethane (50mL) was added Boc₂O (6.57g, 30.1mmol), followed by Et addition₃N (10.4ml, 74.9 mmol). The resulting solution was stirred at 25 ℃ overnight and then concentrated in vacuo. The crude product was purified by FCC, eluting with ethyl acetate/petroleum ether (PE/EA 3: 1) to provide tert-butyl (4-bromophenylethyl) carbamate as a white solid (7.1g, 95%). LCMS (ESI, m/z): 300[ M + H]⁺。

Step 2.4- (4- (2- ((tert-butoxycarbonyl) amino) ethyl) phenyl) piperazine-1-carboxylic acid benzyl ester

To a 100-mL round bottom flask purged and maintained with a nitrogen inert atmosphere was added tert-butyl (4-bromophenylethyl) carbamate (4.00g, 13.3mmol) and anhydrous toluene (50 mL). To the resulting solution was added benzyl piperazine-1-carboxylate (3.53g, 16.0mmol), Pd (OAc)₂(300mg, 1.34mmol), XPhos (1.28g, 2.69mmol), and Cs₂CO₃(13.1g, 40.0 mmol). The reaction mixture was stirred in an oil bath overnight at 105 ℃ and then cooled to room temperature and purified by addition of H₂O (200mL) quench. The resulting mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (1 × 200mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by FCC, eluting with ethyl acetate/petroleum ether (PE/EA ═ 3: 1) to afford benzyl 4- (4- (2- ((tert-butoxycarbonyl) amino) ethyl) phenyl) piperazine-1-carboxylate (5g, 85%) as a yellow solid. LCMS (ESI, m/z): 440[ M + H ]]⁺。

Step 3.4- (4- (2-aminoethyl) phenyl) piperazine-1-carboxylic acid benzyl ester (hydrochloride)

To a 100-mL round bottom flask was added benzyl 4- (4- (2- ((tert-butoxycarbonyl) amino) ethyl) phenyl) piperazine-1-carboxylate (3.0g, 6.83mmol), followed by 4N hydrogen chloride/dioxane (10 mL). The resulting solution was stirred at room temperature for 1h, then concentrated in vacuo to afford benzyl 4- (4- (2-aminoethyl) phenyl) piperazine-1-carboxylate (hydrochloride salt) (2.0g, 86%) as a yellow solid. LCMS (ESI, m/z): 340[ M + H ]]⁺。

Example 4: intermediate 4.4- [4- (2-aminoethyl) -2-chlorophenyl ] piperazine-1-carboxylic acid tert-butyl ester

Step 1 benzyl N- [2- (4-bromophenyl) ethyl ] carbamate

To a 1-L3-necked round bottom flask was added 2- (4-bromophenyl) ethan-1-amine (80.0g, 400mmol), anhydrous DCM (800mL), and TEA (48.7g, 67.1mL, 481 mmol). The resulting mixture was cooled to 0 ℃ and then a solution of benzyl chloroformate (68.3g, 56.9mL, 400mmol) in anhydrous DCM (20mL) was added dropwise. The resulting solution was stirred at 0 ℃ for 2h and then concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 3) to give N- [2- (4-bromophenyl) ethyl ] as a white solid]Benzyl carbamate (100g, 75%). LCMS (ESI, m/z): 334 [ M + H ]]⁺。

Step 2.4- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) phenyl ] piperazine-1-carboxylic acid tert-butyl ester

To a 1-L round bottom flask purged and maintained with a nitrogen inert atmosphere was added N- [2- (4-bromophenyl) ethyl]Benzyl carbamate (50.0g, 150mmol), toluene (500mL), piperazine-1-carboxylic acid tert-butyl ester (34.0 g, 183mmol), Pd (OAc)₂(3.40g, 15.1mmol), XPhos (14.3g, 30.3mmol), and Cs₂CO₃(98.0g, 301 mmol). The resulting mixture was stirred at 105 ℃ overnight, and then cooled to room temperature and poured into water (300 mL). Will be describedThe resulting mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 1) to provide 4- [4- (2- [ [ (benzyloxy) carbonyl ] as a yellow solid]Amino group]Ethyl) phenyl]Piperazine-1-carboxylic acid tert-butyl ester (26g, 40%). LCMS (ESI, m/z): 440[ M + H ]]⁺。

Step 3.4- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) -2-chlorophenyl ] piperazine-1-carboxylic acid tert-butyl ester

To a 250-mL round bottom flask was added 4- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) phenyl]Piperazine-1-carboxylic acid tert-butyl ester (5.40g, 12.3mmol), DCM (100mL), and N-chlorosuccinimide (1.64 g, 12.3 mmol). The resulting solution was stirred at reflux overnight, and then cooled to room temperature and concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 1) to give 4- [4- (2- [ [ (benzyloxy) carbonyl ] as an orange oil]Amino group]Ethyl) -2-chlorophenyl]Piperazine-1-carboxylic acid tert-butyl ester (5.1g, 88%). LCMS (ESI, m/z): 474[ M + H]⁺。

Step 4.4- [4- (2-aminoethyl) -2-chlorophenyl ] piperazine-1-carboxylic acid tert-butyl ester

To a 500-mL round bottom flask purged and maintained under a nitrogen atmosphere were added Raney nickel (5g), methanol (250mL), and 4- [4- (2- [ [ (benzyloxy) carbonyl ] 4]Amino group]Ethyl) -2-chlorophenyl]Piperazine-1-carboxylic acid tert-butyl ester (10.0g, 21.1 mmol). The resulting mixture was bubbled with hydrogen gas, and then stirred at room temperature under a hydrogen atmosphere overnight. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo to provide 4- [4- (2-aminoethyl) -2-chlorophenyl as a gray solid]Piperazine-1-carboxylic acid tert-butyl ester (6.8g, 95%) which was continued without further purification. LCMS (ESI, m/z): 340[ M + H ]]⁺。

Example 5: intermediate 5.3- [4- (2-aminoethyl) -2-chlorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Step 1.3- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) phenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 50-mL 3-necked round-bottomed flask purged and maintained under a nitrogen atmosphere was added N- [2- (4-bromophenyl) ethyl group]Benzyl carbamate (0.692g, 2.07mmol), toluene (10mL), 3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.400g, 1.88mmol), Cs₂CO₃(1.85g，5.68mmol)、 Pd(dppf)Cl₂(0.078g, 0.10mmol), and XPhos (0.090g, 0.20 mmol). The resulting solution was stirred in an oil bath overnight at 100 ℃, and then cooled to room temperature and concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 10) to give 3- [4- (2- [ [ (benzyloxy) carbonyl ] as a yellow oil]Amino group]Ethyl) phenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (440mg, 50%). LCMS (ESI, m/z): 466[ M + H]⁺。

Step 2.3- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) -2-chlorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 25-mL round bottom flask was added 3- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) phenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.44g, 0.95mmol), DCM (5mL), and NCS (0.13g, 0.94 mmol). The resulting solution was stirred in an oil bath overnight at 40 ℃. The reaction mixture was concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 10) to give 3- [4- (2- [ [ (benzyloxy) carbonyl ] as a yellow oil]Amino group]Ethyl) -2-chlorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (200mg, 42%). LCMS (ESI, m/z): 500[ M + H ]]⁺。

Step 3.3- [4- (2-aminoethyl) -2-chlorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 50-mL round bottom flask was added 3- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) -2-chlorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.2)0g, 0.40mmol), 1, 4-dioxane (6mL), and potassium tert-butoxide (0.135g, 1.20 mmol). The resulting solution was stirred in an oil bath for 2h at 100 ℃ and then cooled to room temperature. The reaction mixture was diluted with water (10mL) and then extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to provide 3- [4- (2-aminoethyl) -2-chlorophenyl as an off-white solid]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (140mg, 96%). LCMS (ESI, m/z): 366[ M + H]⁺。

Example 6: intermediate tert-butyl 6.3- (4- (2-aminoethyl) phenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylate

To a 50-mL round bottom flask purged and maintained under nitrogen atmosphere was added 3- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) phenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.25g, 0.54 mmol), methanol (8mL), and 10% palladium on carbon (100 mg). The resulting mixture was bubbled with hydrogen and then stirred at room temperature under a hydrogen atmosphere for 2 h. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo to provide 3- (4- (2-aminoethyl) phenyl) -3, 8-diazabicyclo [3.2.1] as a yellow oil]Octane-8-carboxylic acid tert-butyl ester (170mg, 96%) which was continued without further purification. LCMS (ESI, m/z): 332[ M + H]⁺。

Example 7-1: intermediate tert-butyl 7-1.3- [4- (2-aminoethyl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylate

Step 1.3- (4-bromo-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 1-L round bottom flask purged and maintained with a nitrogen inert atmosphere was added 1,4-dibromo-2, 5-difluorobenzene (15.0g, 55.0mmol), 3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (10.6g, 49.9 mmol), Pd₂(dba)₃CHCl₃(2.59g, 2.50mmol), XantPhos (2.89g, 5.00mmol), sodium tert-butoxide (9.60g, 99.9mmol), and toluene (500 mL). The resulting solution was stirred at 70 ℃ for 45min, and then cooled to room temperature and quenched by the addition of water (100 mL). The resulting solution was extracted with ethyl acetate (3 × 100mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 10) to give 3- (4-bromo-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1] as a pale yellow oil]Octane-8-carboxylic acid tert-butyl ester (15g, 67%). LCMS (ESI, m/z): 403,405[ M + H ]]⁺。

Step 2.3- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 1-L round bottom flask purged and maintained with a nitrogen inert atmosphere was added 3- (4-bromo-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (14.0g, 34.7mmol) and [2- (benzyloxycarbonylamino) ethyl ester]Potassium trifluoroborate (10.9g, 38.2mmol), Pd (dppf) Cl₂(2.55g，3.49 mmol)、RuPhos(3.25g，6.96mmol)、Cs₂CO₃(22.7g, 69.7mmol), toluene (500mL), and water (100 mL). The reaction mixture was stirred at 100 ℃ for 3h and then cooled to room temperature. The resulting mixture was extracted with ethyl acetate (3 × 100mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (PE/EA ═ 100: 1 to 5: 1) to afford 3- [4- (2- [ [ (benzyloxy) carbonyl) as a yellow oil]Amino group]Ethyl) -2, 5-difluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (12g, 62%). LCMS (ESI, m/z): 502[ M + H]⁺。

Step 3.3- [4- (2-aminoethyl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Towards inA1-L round bottom flask purged and maintained under nitrogen atmosphere was charged with 3- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) -2, 5-difluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (12.0 g, 23.9mmol), 10% palladium on carbon (12g), and methanol (500 mL). Subjecting the mixture to hydrogenation with hydrogen₂Bubbled and then stirred at 20 ℃ for 1h under a hydrogen atmosphere. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with methylene chloride/methanol (100: 1 to 10: 1) to provide 3- [4- (2-aminoethyl) -2, 5-difluorophenyl group as a pale yellow oil]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (8.91g, 99%). LCMS (ESI, m/z): 368[ M + H]⁺。

The intermediates in table 1 below were synthesized according to the procedure outlined above for example 7-1, intermediate 7-1, using the appropriate synthesis precursors.

Table 1:

example 8: intermediate tert-butyl 8.3- (4-bromo-2-cyanophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylate

To a 100-mL round-bottom flask was added 3, 8-diazabicyclo [3.2.1]]Octane-8-carboxylic acid tert-butyl ester (1.00g, 4.71mmol), 5-bromo-2-fluorobenzonitrile (1.88g, 9.40mmol), DIEA (1.83g, 2.47mL, 14.2mmol), and DMSO (25 mL). The resulting mixture was stirred in an oil bath at 80 ℃ for 24h, and then cooled to room temperature and quenched by the addition of water (20 mL). The resulting solution was extracted with ethyl acetate (2 × 20 mL). And the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with ethyl acetate/petroleum ether (1: 5) to provide 3- (4-bromo-2-cyanophenyl) -3, 8-diazabicyclo [3.2.1] as a yellow oil]Octane-8-carboxylic acid tert-butyl ester (550mg, 30%). LCMS (ESI, m/z): 392, 394[ M + H ]]⁺。

Example 9: intermediate 9.3- (4-bromo-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Step 1.3- (2-fluoro-4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask purged and maintained with a nitrogen inert atmosphere was added 1, 2-difluoro-4-nitrobenzene (1.00g, 6.29mmol), 3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.47g, 6.92mmol), potassium carbonate (2.60g, 18.8mmol), and DMF (10 mL). The resulting mixture was stirred at 90 ℃ for 4h, and then cooled to room temperature and quenched by the addition of water (20 mL). The resulting solution was extracted with ethyl acetate (3 × 10mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 10) to provide 3- (2-fluoro-4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] as a yellow solid]Octane-8-carboxylic acid tert-butyl ester (1.93g, 87%). LCMS (ESI, m/z): 352[ M + H]⁺。

Step 2.3- (4-amino-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask was added 3- (2-fluoro-4-nitrophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.63g, 4.64mmol), iron powder (1.30g), ammonium chloride (1.23g, 23.0 mmol), tetrahydrofuran (18mL), methanol (18mL), and water (3 mL). The resulting mixture was stirred at 80 ℃ for 4h and then cooled to room temperature. The solids were removed by filtration, and the filtrate was diluted with ethyl acetate (60mL) and washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to provide 3- (4-amino-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] as a yellow oil]Octane-8-carboxylic acid tert-butyl ester (1.71 g). Using the substance without further useAnd (5) purifying. LCMS (ESI, m/z): 322[ M + H]⁺。

Step 3.3- (4-bromo-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round-bottom flask purged and maintained with a nitrogen inert atmosphere was added 3- (4-amino-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.71g, 5.32mmol), acetonitrile (30mL), CuBr (1.53g, 10.7mmol), and isoamyl nitrite (0.938g, 8.02 mmol). The resulting mixture was stirred at 60 ℃ for 3h, and then cooled to room temperature and concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 8) to give 3- (4-bromo-2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] as a yellow oil]Octane-8-carboxylic acid tert-butyl ester (448mg, 22%). LCMS (ESI, m/z): 385, 387[ M + H ]]⁺。

The intermediates in table 2 below were synthesized according to the procedure outlined above for example 7-1, intermediate 7-1 (steps 2 and 3) using the appropriate synthesis precursors.

Table 2:

example 11-1: intermediate 11-1.3- [4- (2-aminoethyl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Step 1.2- (4-bromo-2-fluorophenyl) ethan-1-amine

To a 100-mL round bottom flask were added 2- (4-bromo-2-fluorophenyl) acetonitrile (1.20g, 5.61mmol), borane tetrahydrofuran complex (1M; 16.8mL, 16.8mmol), and THF (20 mL). The resulting solution was stirred in an oil bath for 18h at 70 ℃ and then cooled to room temperature and quenched by the addition of methanol (5 mL). The resulting mixture was concentrated in vacuo to provide 2- (4-bromo-2-fluorophenyl) ethan-1-amine (900mg, 74%) as a brown oil, which was not requiredFurther purification is continued. LCMS (ESI, m/z): 218, 220 [ M + H [ ]]⁺。

Step 2 benzyl N- [2- (4-bromo-2-fluorophenyl) ethyl ] carbamate

To a 100-mL round bottom flask was added 2- (4-bromo-2-fluorophenyl) ethan-1-amine (0.900g, 4.13mmol), benzyl chloroformate (0.915g, 0.763mL, 5.37mmol), triethylamine (1.25g, 1.72mL, 12.38 mmol), and dichloromethane (10 mL). The resulting solution was stirred at 20 ℃ for 2h, and then quenched by addition of water (10mL) and extracted with ethyl acetate (3 × 20 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via silica gel chromatography and eluted with ethyl acetate/petroleum ether (PE/EA ═ 100: 1 to 10: 1) to afford N- [2- (4-bromo-2-fluorophenyl) ethyl ester as a yellow solid]Benzyl carbamate (1.2g, 83%). LCMS (ESI, m/z): 352, 354 [ M + H ]]⁺。

Step 3.8- [4- (2- [ [ (benzyloxy) carbonyl ] amino ] ethyl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-3-carboxylic acid tert-butyl ester

To a 50-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was added N- [2- (4-bromo-2-fluorophenyl) ethyl ] ethyl]Benzyl carbamate (0.500g, 1.42mmol), 3, 8-diazabicyclo [3.2.1]Octane-3-carboxylic acid tert-butyl ester (0.320g, 1.51mmol), Cs₂CO₃(1.40g，4.30mmol)、Pd(OAc)₂(0.032g, 0.14mmol), RuPhos (0.13g, 0.29mmol), and toluene (10 mL). The resulting mixture was stirred in an oil bath for 2h at 100 ℃ and then cooled to room temperature and quenched by the addition of water (20 mL). The resulting solution was extracted with ethyl acetate (3 × 20mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (PE/EA ═ 100: 1 to 3: 1) to afford 8- [4- (2- [ [ (benzyloxy) carbonyl) as a pale yellow oil]Amino group]Ethyl) -3-fluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-3-carboxylic acid tert-butyl ester (220mg, 32%). LCMS (ESI, m/z): 484[ M + H]⁺。

Step 4.3- [4- (2-aminoethyl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 50-mL round bottom flask purged and maintained under a nitrogen atmosphere was added 8- [4- (2- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Ethyl) -3-fluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-3-carboxylic acid tert-butyl ester (0.22g, 0.45mmol), and methanol (10mL), followed by addition of 10% palladium on carbon (220 mg). The reaction mixture was bubbled with hydrogen and stirred at 20 ℃ under a hydrogen atmosphere (balloon pressure) for 2 h. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo to provide 3- [4- (2-aminoethyl) -3-fluorophenyl ] as a pale yellow oil]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (70mg, 40%), which was continued without further purification. LCMS (ESI, m/z): 350[ M + H ]]⁺。

The intermediates in table 3 below were synthesized according to the procedure outlined above for example 11-1, intermediate 11-1, using the appropriate synthesis precursors.

Table 3:

example 12-1: intermediate tert-butyl 12-1.3- [4- (1-aminopropan-2-yl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylate

Step 1.3- (4-acetyl-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 250-mL round bottom flask purged and maintained with a nitrogen inert atmosphere was added 3- (4-bromo-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.30g, 3.22mmol), tributyl (1-ethoxyvinyl) stannane (1.75g, 1.64mL, 4.85mmol), Pd (dppf) Cl₂CH₂Cl₂(0.53 g, 0.64mmol), DIEA (1.25g, 1.68mL, 9.67mmol), and toluene (100 mL). The resulting solution was stirred at 100 ℃ for 18h, and then cooled to room temperature and purified by additionSaturated aqueous ammonium chloride solution (30mL) was quenched. The resulting mixture was extracted with ethyl acetate (3 × 100mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with ethyl acetate/petroleum ether (1: 100 to 1: 10) to provide 3- (4-acetyl-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1] as a pale yellow oil]Octane-8-carboxylic acid tert-butyl ester (460mg, 35%). LCMS (ESI, m/z): 367[ M + H]⁺。

Step 2.3- [4- (1-cyanoethyl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask was added 3- (4-acetyl-2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1]Tert-butyl octane-8-carboxylate (0.420g, 1.15mmol), p-toluenesulfonyl isocyanide (0.336 g, 1.71mmol), potassium tert-butoxide (0.321g, 2.86mmol), tert-butanol (10mL), and ethylene glycol dimethyl ether (10 mL). The resulting solution was stirred at 90 ℃ for 18h, and then cooled to room temperature and quenched by the addition of water (50 mL). The resulting mixture was extracted with ethyl acetate (3 × 20mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 100 to 1: 10) to give 3- [4- (1-cyanoethyl) -2, 5-difluorophenyl group as a pale yellow solid]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (350mg, 73%). LCMS (ESI, m/z): 378[ M + H]⁺。

Step 3.3- [4- (1-aminopropan-2-yl) -2, 5-difluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 50-mL round bottom flask purged and maintained under a nitrogen atmosphere was added 3- [4- (1-cyanoethyl) -2, 5-difluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.35g, 0.93 mmol), Raney nickel (0.350g), and a solution of ammonia in methanol (7M, 20 mL). Subjecting the mixture to hydrogenation with hydrogen₂Bubbled and then stirred under a hydrogen atmosphere (balloon pressure) at 20 ℃ for 2 h. The solids were removed by filtration through Celite and the filtrate was concentrated in vacuo to provide 3- [4- (1-Aminopropan-2-yl) -2, 5-difluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (200mg, 51%). LCMS (ESI, m/z): 382[ M + H ]]⁺。

The intermediates in table 4 below were synthesized according to the procedure outlined above for example 12-1, intermediate 12-1, using the appropriate synthesis precursors.

Table 4:

example 13-1: intermediate 13-1.3- [4- (1-aminopropan-2-yl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Step 1.3- (4-acetyl-3-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round-bottomed flask were added 1- (2, 4-difluorophenyl) ethan-1-one (2.65g, 17.0mmol), 3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (3.00g, 14.1mmol), potassium carbonate (5.86 g, 42.4mmol), and HMPA (30 mL). The resulting solution was stirred in an oil bath overnight at 70 ℃, and then cooled to room temperature and quenched by the addition of water (30 mL). The resulting solution was extracted with ethyl acetate (3 × 30mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 5) to give 3- (4-acetyl-3-fluorophenyl) -3, 8-diazabicyclo [3.2.1] as a brown oil]Octane-8-carboxylic acid tert-butyl ester (1.8g, 30%). LCMS (ESI, m/z): 349[ M + H]⁺。

Step 2.3- [4- (1-cyanoethyl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask was added 3- (4-acetyl-3-fluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.80g,5.17mmol), KOt-Bu (1.45g, 13.0mmol), p-toluenesulfonyl isocyanide (1.51g, 7.74mmol), tert-butyl ester (20mL), and ethylene glycol dimethyl ether (20 mL). The resulting solution was stirred in an oil bath overnight at 90 ℃ and then cooled and quenched by the addition of water (20 mL). The resulting solution was extracted with ethyl acetate (3 × 20mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by chromatography on silica gel and eluted with ethyl acetate/petroleum ether (1: 5) to give 3- [4- (1-cyanoethyl) -3-fluorophenyl ] ether as a brown oil]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.2g, 65%). LCMS (ESI, m/z): 360[ M + H ]]⁺。

Step 3.3- [4- (1-aminopropan-2-yl) -3-fluorophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask purged and maintained under nitrogen atmosphere was added 3- [4- (1-cyanoethyl) -3-fluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.00g, 2.78mmol), NH in MeOH₃(7M, 20mL), and Raney nickel (500 mg). The reaction mixture was bubbled with hydrogen and stirred at room temperature under a hydrogen atmosphere (balloon pressure) for 2 h. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo. The crude product was purified by chromatography on silica gel eluting with methylene chloride/methanol (10: 1) to provide 3- [4- (1-aminopropan-2-yl) -3-fluorophenyl ] as a yellow oil]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (800mg, 79%). LCMS (ESI, m/z): 364[ M + H ]]⁺。

The intermediates in table 5 below were synthesized according to the procedure outlined above for example 13-1, intermediate 13-1, using the appropriate synthesis precursors.

Table 5:

example 14: intermediate 14.3- (4- (1-aminopropan-2-yl) -2-cyanophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Step 1.3- (4-acetyl-2-bromophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 20-mL microwave tube was added 1- (3-bromo-4-fluorophenyl) ethan-1-one (3.00g, 13.8mmol), 3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (3.67g, 17.3mmol), DIEA (5.48g, 7.39mL, 42.4mmol), and DMSO (8 mL). The resulting solution was heated at 120 ℃ for 4h under microwave irradiation. The reaction mixture was cooled to room temperature and then poured into water (30 mL). The resulting mixture was extracted with ethyl acetate (3 × 30mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with ethyl acetate/petroleum ether (1: 5) to provide 3- (4-acetyl-2-bromophenyl) -3, 8-diazabicyclo [3.2.1] as a yellow oil]Octane-8-carboxylic acid tert-butyl ester (1.5g, 27%). LCMS (ESI, m/z): 409, 411[ M + H]⁺。

Step 2.3- [ 2-bromo-4- (1-cyanoethyl) phenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere, potassium tert-butoxide (0.480g, 4.28mmol) and THF (15mL) were added followed by dropwise addition of a solution of p-toluenesulfonyl isocyanide (0.500g, 2.56mmol) in tetrahydrofuran (3mL) with stirring at-78 ℃. The resulting solution was stirred at-78 ℃ for 15 minutes and then added dropwise at-78 ℃ with stirring 3- (4-acetyl-2-bromophenyl) -3, 8-diazabicyclo [3.2.1] at-78 ℃ with stirring]A solution of tert-butyl octane-8-carboxylate (0.700g, 1.71mmol) in tetrahydrofuran (5 mL). The resulting mixture was then stirred at this temperature for a further 1.5 h. Methanol (10mL) was added and the resulting solution was heated to 80 ℃ and stirred for an additional 30 minutes. The reaction was then cooled to room temperature and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with ethyl acetate/petroleum ether (1: 5) to provide 3- [ 2-bromo-4- (1-cyanoethyl) as a yellow oil) Phenyl radical]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (500mg, 70%). LCMS (ESI, m/z): 420, 422[ M + H]⁺。

Step 3.3- [4- (1-aminopropan-2-yl) -2-bromophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 250-mL round bottom flask was added 3- [ 2-bromo-4- (1-cyanoethyl) phenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.50g, 3.57mmol) and borane tetrahydrofuran complex (1M, 20mL, 20 mmol). The resulting solution was stirred at 25 ℃ for 2h and then quenched by the addition of methanol (30 mL). The resulting mixture was concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with methylene chloride/methanol (10: 1) to provide 3- [4- (1-aminopropan-2-yl) -2-bromophenyl group as a white solid]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.3g, 86%). LCMS (ESI, m/z): 424, 426[ M + H]⁺。

Step 4.3- [4- (1- [ [ (benzyloxy) carbonyl ] amino ] propan-2-yl) -2-bromophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask was added 3- [4- (1-aminopropan-2-yl) -2-bromophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.40g, 3.30mmol), potassium carbonate (1.37g, 9.91mmol), ethyl acetate (20mL), water (20mL), and CbzCl (0.619g, 0.516mL, 3.63 mmol). The resulting mixture was stirred in an oil bath for 3h at 60 ℃ and then cooled to room temperature. The reaction mixture was diluted with water (100mL) and then extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by silica gel chromatography and eluted with ethyl acetate/petroleum ether (1: 1) to give 3- [4- (1- [ [ (benzyloxy) carbonyl) as a pale yellow oil]Amino group]Propan-2-yl) -2-bromophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (1.0g, 54%). LCMS (ESI, m/z): 558, 560[ M + H ]]⁺。

Step 5.3- [4- (1- [ [ (benzyloxy) carbonyl ] amino ] propan-2-yl) -2-cyanophenyl ] -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 10-mL microwave tube purged and maintained with a nitrogen inert atmosphere was placed a 3- [4- (1- [ [ (benzyloxy) carbonyl ] carbonyl group]Amino group]Propan-2-yl) -2-bromophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.800mg, 1.43mmol), Zn (CN)₂(0.167g，1.42mmol)、Pd(PPh₃)₄(0.166g, 0.14mmol), and DMF (3 mL). The resulting mixture was heated at 120 ℃ for 4h under microwave irradiation. The reaction mixture was then cooled to room temperature, poured into water (50mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were concentrated in vacuo and the crude product was purified via preparative TLC plates and eluted with ethyl acetate/petroleum ether (1: 3) to afford 3- [4- (1- [ [ (benzyloxy) carbonyl) as a colorless oil]Amino group]Propan-2-yl) -2-cyanophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (400 mg, 55%). LCMS (ESI, m/z): 505[ M + H]⁺。

Step 6.3- (4- (1-aminopropan-2-yl) -2-cyanophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 100-mL round bottom flask purged and maintained under a nitrogen atmosphere was added 3- [4- (1- [ [ (benzyloxy) carbonyl ] carbonyl]Amino group]Propan-2-yl) -2-cyanophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.35g, 0.69mmol), tetrahydrofuran (15mL), and 10% palladium on carbon (350 mg). The reaction mixture was bubbled with hydrogen and stirred at room temperature under a hydrogen atmosphere (balloon pressure) for 1 h. The solids were removed by filtration through celite, and the filtrate was concentrated in vacuo. The crude product was purified via preparative TLC plate and eluted with methylene chloride/methanol (10: 1) to afford 3- (4- (1-aminopropan-2-yl) -2-cyanophenyl) -3, 8-diazabicyclo [3.2.1] as a brown oil]Octane-8-carboxylic acid tert-butyl ester (180mg, 70%). LCMS (ESI, m/z): 371[ M + H]⁺。

Process for the synthesis of compounds of formula (I)

Example 15-1 (I-1): 7-amino-3-methyl-N- (4- (piperazin-1-yl) phenethyl) thieno [2,3-b ] pyrazine-6-carboxamide

Step 1.4- (4- (2- (7-amino-3-methylthio-thieno [2,3-b ] pyrazine-6-carboxamide) ethyl) phenyl) piperazine-1-carboxylic acid benzyl ester

To an 8-mL vial was added 7-amino-3-methylthioeno [2,3-b]Pyrazine-6-carboxylic acid (0.11g, 0.53 mmol), 4- [4- (2-aminoethyl) phenyl]Piperazine-1-carboxylic acid benzyl ester hydrochloride (0.259g, 0.58mmol), HATU (0.240g, 0.63mmol), DMF (5mL), and DIEA (0.2g, 0.3mL, 1.72 mmol). The resulting solution was stirred at room temperature overnight. The solids were removed by filtration and the filtrate was purified by preparative HPLC using the following conditions: (Waters I) column: XBridge C18, 19x150mm, 5 μm; mobile phase, phase a: water (0.05% NH)₄OH); phase B: CH (CH)₃CN (30% up to 85 in 7 min); flow rate: 20 mL/min; detector, 254 nm. This provided 4- (4- (2- (7-amino-3-methylthioeno [2, 3-b) ] as a yellow solid]Pyrazine-6-carboxamide) ethyl) phenyl) piperazine-1-carboxylic acid benzyl ester (120mg, 43%). LCMS (ESI, m/z): 531[ M + H]⁺。¹H NMR(300MHz，DMSO-d₆)：δppm8.64(s， 1H)，7.93(t，J＝5.7Hz，1H)，7.41-7.30(m，5H)，7.29-7.08(m，2H)，6.91-6.88 (m，4H)，5.10(s，2H)，3.53-3.50(m，4H)，3.42-3.31(m，2H)，3.09-3.06(m，4H)， 2.76-2.71(m，2H)，2.64(s，3H)。

Step 2.7-amino-3-methyl-N- (4- (piperazin-1-yl) phenethyl) thieno [2,3-b ] pyrazine-6-carboxamide

To a 50-mL round bottom flask was added 4- (4- (2- (7-amino-3-methylthio-eno [2,3-b ]]Pyrazine-6-carboxamide) ethyl) phenyl) piperazine-1-carboxylic acid benzyl ester (0.050g, 0.09mmol), and dichloromethane (10 mL). The resulting solution was cooled to-20 ℃ and BBr was added dropwise₃(1M in DCM; 1mL, 1 mmol). The reaction mixture was stirred at-20 ℃ for 2h and then quenched by the addition of methanol (10 mL). The resulting mixture was concentrated in vacuo, diluted with DMF (4mL) and purified by preparative HPLC using the following conditions: column: XBridge C18, 19x150mm, 5 μm; a mobile phase; phase A: water (10mm NH)₄HCO₃+0.05％NH₄OH); phase B: CH (CH)₃CN (20% to 70% in 8 min)(ii) a Flow rate: 20 mL/min; detector wavelength: 254 nm. This provided 7-amino-3-methyl-N- (4- (piperazin-1-yl) phenethyl) thieno [2,3-b as a yellow solid]Pyrazine-6-carboxamide (28mg, 74%). LCMS (ESI, m/z): 391[ M + H]⁺。¹H NMR(300MHz，DMSO-d₆)：6ppm 8.64(s，1H)，7.96-7.92(m， 1H)，7.09-701(m，1H)，6.88-6.94(m，4H)，3.65-3.37(m，2H)，2.99-2.97(m， 4H)，2.91-2.71(m，6H)，2.65(s，3H)。

The examples in Table 6 below were synthesized according to the procedure outlined above for example 15-1(I-1) using the appropriate synthetic precursors. Additional details surrounding the synthetic methods and HPLC purification conditions appear in the examples below.

Table 6:

example 16-1 (I-3): n- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-chlorophenethyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide (hydrochloride)

Step 1.3- (4- (2- (7-amino-3-methylthio-thieno [2,3-b ] pyrazine-6-carboxamide) ethyl) -2-chlorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 25-mL round bottom flask was added 3- [4- (2-aminoethyl) -2-chlorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.100g, 0.27mmol), dichloromethane (5mL), 7-amino-3-methylthioeno [2,3-b ]]Pyrazine-6-carboxylic acid (0.058g, 0.28mmol), EDCI (0.063g, 0.33mmol), HOBT (0.040g, 0.30mmol), and DIEA (0.106g, 0.143mL, 0.82 mmol). The resulting solution was stirred in an oil bath for 2h at 40 ℃. The reaction mixture was concentrated in vacuo, and the crude product was dissolved in DMF (2mL) and purified by preparative HPLC using the following conditions: column: SunAire Prep C185 μm 19x150 mm; mobile phase: water (W)(containing 0.1% formic acid) and CH₃CN (CH within 6 min)₃CN 35% up to 65%); detector wavelength: 254 nm. This provided 3- (4- (2- (7-amino-3-methylthioeno [2, 3-b) ] as a yellow solid]Pyrazine-6-carboxamide) ethyl) -2-chlorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (50mg, 33%). LCMS (ESI, m/z): 557[ M + H ]]⁺。

Step 2.N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-chlorophenylethyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide (hydrochloride)

To a 25-mL round bottom flask was added 3- (4- (2- (7-amino-3-methylthio-eno [2,3-b ]]Pyrazine-6-carboxamide) ethyl) -2-chlorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.040g, 0.07 mmol), methanol (1mL), and 4N HCl/dioxane (2 mL). The resulting solution was stirred at room temperature for 2h, and then concentrated in vacuo. The solid was triturated with ether/MeOH (10: 1; 10mL) and collected by filtration to provide N- (4- (3, 8-diazabicyclo [3.2.1] as a yellow solid]Octane-3-yl) -3-chlorophenethyl) -7-amino-3-methylthio [2,3-b]Pyrazine-6-carboxamide (hydrochloride; 16.9mg, 48%). LCMS (ESI, m/z): 493[ M + H ]]⁺。¹H NMR(300MHz，MeOD)：δppm 8.59(s，1H)，7.38(s， 1H)，7.25-7.21(m，1H)，7.16-7.12(m，1H)，4.12(br s，2H)，3.68-3.52(m，2H)， 3.33-3.29。(m，2H)，3.19-3.15(m 2H)，2.90-2.86(m，2H)，2.70(s，3H)。2.46-2.41 (m，2H)，2.11-2.08(m，2H)。

The examples in Table 7 below were synthesized according to the procedure outlined above for example 16-1(I-3) using the appropriate synthesis precursors.

Table 7:

example 17-1 (I-7): n- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 5-difluorophenethyl) -7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxamide

Step 1.3- (4- (2- (7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide) ethyl) -2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

To a 500-mL round bottom flask was added 3- [4- (2-aminoethyl) -2, 5-difluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (3.00g, 8.16mmol), 7-amino-3-methylthioeno [2,3-b ]]Pyrazine-6-carboxylic acid (2.05g, 9.80mmol), EDCI (2.04g, 10.64mmol), HOBT (1.32 g, 9.77mmol), DIEA (3.17g, 4.27mL, 24.5mmol), and DMF (100 mL). The resulting solution was stirred at 20 ℃ for 1h and then poured into water (500 mL). The resulting precipitate was collected by filtration and dried in vacuo to provide 3- (4- (2- (7-amino-3-methylthiophene [2, 3-b) ] as a yellow solid]Pyrazine-6-carboxamide) ethyl) -2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (2.4g, 50%). LCMS (ESI, m/z): 559[ M + H ]]⁺。

Step 2.N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 5-difluorophenethyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide

To a 100-mL round bottom flask was added 3- (4- (2- (7-amino-3-methylthio-eno [2, 3-b))]Pyrazine-6-carboxamide) ethyl) -2, 5-difluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (2.80g, 5.01mmol), dichloromethane (50mL), and trifluoroacetic acid (10 mL). The resulting solution was stirred at 20 ℃ for 1h and then concentrated in vacuo. The crude product was slurried with a solution of ammonia in methanol (7M; 50 mL). The solids were removed by filtration and the filtrate was concentrated in vacuo to afford the crude product, which was passed through C₁₈Purification by reverse phase silica gel chromatography and purification with acetonitrile and aqueous ammonium bicarbonate solution (10mmol/L NH)₄HCO₃) (from 0-60%) elution was performed. This provided N- (4-,(s) as a yellow solid3, 8-diazabicyclo [3.2.1]Octane-3-yl) -2, 5-difluorophenethyl) -7-amino-3-methylthiophene [2,3-b]Pyrazine-6-carboxamide (1.5g, 65%). LCMS (ESI, m/z): 459[ M + H ]]⁺。¹H NMR(300MHz， DMSO-d₆)δppm：8.64(s，1H)，7.96-8.00(m，1H)，6.99-7.06(m，1H)，6.89(s， 2H)，6.65-6.72(m，1H)，3.55-3.63(m，4H)，3.06-3.10(m，2H)，2.74-2.79(m， 4H)，2.64(s，3H)，2.24(br s，1H)，1.74-1.81(m，2H)，1.64-1.71(m，2H)。

The examples in Table 8 below were synthesized according to the procedure outlined above for example 17-1(I-7) using the appropriate synthetic precursors. Additional details surrounding the synthetic methods and HPLC purification conditions appear in the examples below.

Table 8:

examples 18-1A (I-12) and 18-1B (I-13): n- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide (assumed stereochemical configuration) and N- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide (assumed stereochemical configuration)

Step 1.3- (4- ((S) -1- (7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide) propan-2-yl) -2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (assumed stereochemical configuration) and 3- (4- ((R) -1- (7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide) propan-2-yl) -2-fluorophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (assumed stereochemical configuration)

To a 100-mL round bottom flask was added 3- [4- (1-aminopropan-2-yl) -2-fluorophenyl]-3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acid tert-butyl ester (0.120g, 0.33mmol), 7-amino-3-methylthioeno [2,3-b ]]Pyrazine-6-carboxylic acid (0.069g, 0.33mmol), EDCI (0.082g, 0.43mmol), HOBt (0.053 g, 0.39mmol), DIEA (0.085g, 0.66mmol), and DMF (5 mL). The resulting solution was stirred at 20 ℃ overnight and then quenched by the addition of water (10 mL). The resulting mixture was extracted with ethyl acetate (3 × 20mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified via silica gel column chromatography eluting with ethyl acetate/petroleum ether (PE/EA ═ 1: 3). Chiral separation of the racemate by chiral-preparative HPLC using the following conditions: the instrument name: SHIMADZU LC-20AD, LC parameters: pump mode: binary gradient, initial concentration of pump B: 30.0%, total flow: 20mL/min, phase A: hexane (0.1% DEA); phase B: IPA-HPLC; column name: DAICEL CHIRALPAK IA, length: 25mm, inner diameter: 2mm, particle size: 5 μm, column temperature: 20 ℃, PDA type: SPD-M20A, wavelength: from 190nm to 500 nm. This provides: step 1, enantiomer a: the first eluting peak (retention time 22.9min, 27mg (15%)) was a yellow solid. LCMS (ESI, m/z): 555[ M + H ]]⁺(ii) a And step 1, enantiomer B: the second eluting peak (retention time 24.9min, 50mg (27%)) was a yellow solid. LCMS (ESI, m/z): 555[ M + H ]]⁺。

Step 2, example 18-1A (I-12): n- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide (postulated stereochemical configuration)

To a 25-mL round bottom flask was added 3- (4- ((S) -1- (7-amino-3-methylthioeno [2, 3-b)]Pyrazine-6-carboxamide) propan-2-yl) -2-fluorophenyl) -3, 8-diazabicyclo [3.2.1]Octane-8-carboxylic acidTert-butyl ester (enantiomer A, assumed stereochemical configuration; 0.027g, 0.05mmol, 1.00 equiv.), dichloromethane (2mL), and trifluoroacetic acid (0.5 mL). The resulting solution was stirred at 20 ℃ for 30min and then concentrated in vacuo. The resulting crude product was dissolved in DCM (5mL) and washed with NH₃MeOH (7M) adjusted the pH of the solution to about 8. The resulting mixture was concentrated in vacuo and the crude product was purified by preparative HPLC using the following conditions: the instrument name: SHIMADZU LC-20AD, LC parameters: pump mode: binary gradient, initial concentration of pump B: 25.0%, end concentration of pump B: 42.0% total flow: 20mL/min, time: 8min, phase A: water (10mmol/L NH)₄HCO₃) And (2) phase B: MeCN-HPLC, column name: XBridge Prep C18OBD column, length: 150mm, inner diameter: 19mm, particle size: 5 μm, pore size:

column temperature: 25 ℃, PDA type: SPD-M20A, wavelength: from 190nm to 500 nm. This provided N- ((2S) -2- (4- (3, 8-diazabicyclo [ 3.2.1) as a yellow solid]Octane-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthio [2,3-b]Pyrazine-6-carboxamide (assumed stereochemical configuration, 12 mg, 53%). LCMS (ESI, m/z): 455[ M + H]⁺。¹H NMR(400MHz，CD₃OD)：δppm 8.56(s，1H)，6.85-7.02(m，3H)，3.65(s，2H)，3.37-3.48(m，2H)，3.22(d，J＝ 11.2Hz，2H)，2.93-3.10(m，3H)，2.67(s，3H)，2.02-2.07(m，2H)，1.80-1.90 (m，2H)，1.26(d，J＝6.8Hz，3H)。

Step 2, example 18-1B (I-13): n- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide (postulated stereochemical configuration)

Example 18-1B (I-13) was prepared according to the procedure outlined above for example 18-1A (I-12) enantiomer B from step 1. This provided the title compound as a yellow solid (10mg, 25%). LCMS (ESI, m/z): 455[ M + H]⁺。¹H NMR(400MHz，CD₃OD)δ8.56(s，1H)，6.85-7.02 (m，3H)，3.63(s，2H)，3.37-3.48(m，2H)，3.17-3.25(m，2H)，3.01-3.05(m， 1H)，2.96(d，J＝11.3Hz，2H)，2.67(s，3H)，1.98-2.06(m，2H)，1.82-1.88(m， 2H)，1.26(d，J＝7.2Hz，3H)。

The examples in Table 9 below were synthesized according to the procedure outlined above for examples 18-1A (I-12) and 18-1B (I-13) using the appropriate synthesis precursors. Additional details around the synthetic method and chiral HPLC (after step 1) and preparative HPLC purification conditions appear in each of the enantiomeric pairs below.

Table 9:

example 19: biochemical test: ubiquitin-rhodamine 110 assay for USP28 Activity

In a final volume of 20. mu.L, in a medium containing 20mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH8.0 solution; Corning 46-031-CM)), 2mM CaCl₂Each assay was performed in assay buffer (1M calcium chloride solution; Sigma #21114), 2mM BME (2-mercaptoethanol; Sigma 63689-25mL-F), 0.01% Prion (0.22. mu.M filtered, Sigma # G-0411), and 0.01% Triton X-100. Stock compound solutions at-20 ℃ in 10mM DMSO. For up to 1 month prior to assay, 2mM test compound was pre-dispensed into assay plates (black, low volume; Corning # 3820) and frozen at-20 ℃. The pre-printed test panels were allowed to reach room temperature on the day of testing. For screening, 100nL of 2mM pre-allocated to the finalScreening concentration 10. mu.M (DMSO)_(fc)0.5%). The enzyme (USP28), construct USP28(USP28-5(1-1077) -TEV-6 His; Lifesensors) concentration and incubation time were optimized for maximum signal to background ratio while maintaining initial velocity conditions at fixed substrate concentrations. The final concentration of enzyme in the assay was 400 pM. In [ Ub-Rh110]In the case of & lt Km, the final substrate (Ub-Rh 110; ubiquitin-rhodamine 110, R)&D Systems # U-555) at a concentration of 25 nM. 10 μ L of 2 Xenzyme was added to a test plate (pre-printed with compound) either with 2 XUb-Rh 110 or pre-incubated with USP28 for 40 minutes prior to adding 10 μ L of 2 XUb-Rh 110 to the compound plate for 40 minutes. The plates were incubated in stacks at room temperature for 90 minutes before reading the fluorescence either on Envision (excitation at 485nm and emission at 535 nm; Perkin Elmer) or on pherasar (excitation at 485nm and emission at 535 nm; BMG Labtech).

For subsequent studies, each assay was performed in 15 μ L final volume in assay buffer containing 20mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH8.0 solution; Corning 46-031-CM)), 3mM BME (2-mercaptoethanol; Sigma 63689-25mL-F), 0.03% BGG (0.22 μ M filtered, Sigma, G7516-25G), and 0.01% Triton X-100(Sigma, T9284-10L). For final assay concentrations of 25 μ M to 11nM or 25 μ M to 1.3nM, respectively, nanoliter amounts of 8-or 10-point, 3-fold serial dilutions in DMSO were pre-dispensed into assay plates (Perkin Elmer, ProxiPlate-384F Plus, #). The enzyme USP28, construct USP28(USP28-5(1-1077) -TEV-6 × His; LifeSensors) concentration and incubation time were optimized for maximum signal to background ratio while maintaining initial velocity conditions at fixed substrate concentrations. The final concentration of enzyme in the assay was 75 pM. In the case of [ Ub-Rh110] < Km, the final substrate (Ub-Rh 110; ubiquitin-rhodamine 110, R & D Systems # U-555) concentration was 25 nM. mu.L of 2 Xenzyme was added to the assay plate preincubated with USP28 for 30 minutes (preprinted with compound) and then 5. mu.L of 2 XUb-Rh 110 was added to the assay plate. Plates were incubated in stacks at room temperature for 20 minutes, after which 5. mu.L of stop solution (final concentration 10mM citric acid (Sigma, 251275-500G)) was added. Fluorescence was read either on Envision (excitation at 485nm and emission at 535 nm; Perkin Elmer) or on pherasar (excitation at 485nm and emission at 535 nm; BMG Labtech).

Example 20: biochemical test: ubiquitin-rhodamine 110 assay for USP25 Activity

The assay was performed in a final volume of 9. mu.L in assay buffer containing 20mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH8.0 solution; Coming 46-031-CM)), 3mM BME (2-mercaptoethanol; Sigma 63689-25mL-F), 0.03% BGG (0.22. mu.M filtered, Sigma, G7516-25G), and 0.01% Triton X-100(Sigma, T9284-10L). For final assay concentrations of 25 μ M to 1.3nM (up to the lowest dose), respectively, nanoliter amounts of 10-point, 3-fold serial dilutions in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724 BC). The enzyme USP25, construct USP25-His6(Boston Biochem E-546) concentration and incubation time were optimized for maximum signal to background ratio while maintaining the initial velocity conditions at a fixed substrate concentration. The final concentration of enzyme in the assay was 75 pM. In the case of [ Ub-Rh110] < Km, the final substrate (Ub-Rh 110; ubiquitin-rhodamine 110, R & D Systems # U-555) concentration was 25 nM. mu.L of 2 Xenzyme was added to the assay plate preincubated with USP25 for 30 minutes (preprinted with compound) and then 3. mu.L of 2 XUb-Rh 110 was added to the assay plate. Plates were incubated at room temperature for 45 minutes, after which 3. mu.L of stop solution (final concentration 10mM citric acid (Sigma, 251275-500G)) was added. Fluorescence was read either on Envision (excitation at 485nm and emission at 535 nm; Perkin Elmer) or on pherasar (excitation at 485nm and emission at 535 nm; BMG Labtech).

For both test formats, USP28 and USP25, the data are reported as percent inhibition compared to control wells based on the following equation: inhibition%_{Is low in}) /(average)_{Height of}-average_{Is low in}) Where FLU ═ measured fluorescence, average_{Is low in}Mean fluorescence of no enzyme control (n-16) and mean_{Height of}Mean fluorescence for DMSO control (n 16). Determination of IC by Curve fitting of the Standard 4-parameter logistic fitting Algorithm included in the Activity Base software Package (IDBS XE Designer Model205)₅₀The value is obtained. The data were fitted using the Levenburg Marquardt algorithm.

Table 10: the compounds of the present disclosure have USP28 and USP25 activity in USP28 and USP25 assays. + +++ refers to an IC of less than about 0.2 μ M₅₀And +++ represents an IC of between about 0.2 μ M and about 2 μ M₅₀And ++ represents an IC of between about 2 μ M and about 10 μ M₅₀And + represents an IC between about 10 μ M and about 25 μ M₅₀. ND means data not yet determined.

Table 10: USP28 and USP25 tests

Equivalents of

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed within the scope of the following claims.

Claims (17)

1. A compound of formula (I):

or a pharmaceutically acceptable salt or tautomer thereof,

wherein:

x is N or CR₆；

R₁Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl, -CN or-NR₈R₉；

R₂Is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₁-C₆) Haloalkyl, (C)₁-C₆) Haloalkoxy, (C)₁-C₆) Hydroxyalkyl, halogen, (C)₃-C₈) Cycloalkyl or-NR₁₀R₁₁；

R₃Is H, (C)₁-C₆) Alkyl or (C)₁-C₆) A haloalkyl group;

R₄is H, (C)₁-C₆) Alkyl, halogen or (C)₁-C₆) A haloalkyl group;

R_4'is H, (C)₁-C₆) Alkyl, halogen or (C)₁-C₆) A haloalkyl group;

R₅is- (C)₀) Alkylene-piperazinyl;

each R₆Independently at each occurrence is H, halogen or-CN;

each R₈、R₉、R₁₀And R₁₁Independently H, (C)₁-C₆) Alkyl or-C (O) (C)₁-C₆) An alkyl group; and is

n is 0, 1, 2 or 3.

2. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₁Is (C)₁-C₆) Alkyl and R₂Is H.

3. The compound of claim 1, having the structure of formula (Ig):

or a pharmaceutically acceptable salt or tautomer thereof.

4. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₃、R₄And R_4'Each independently selected from H or (C)₁-C₆) An alkyl group.

5. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₆Is halogen or H.

6. The compound of claim 5, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₆Is halogen and n is 2.

7. The compound of claim 3, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₁Is (C)₁-C₆) An alkyl group; r₃And R₄Each independently selected from H or (C)₁-C₆) An alkyl group; r₆Is halogen and n is 2.

8. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein X is CH.

9. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₃Is H or CH₃。

10. The compound of claim 7, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₄Is H or CH₃And R is_4'Is H.

11. The compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof, wherein X is CH; r₃Is H or CH₃；R₄Is H or CH₃(ii) a And R is_4'Is H.

12. The compound of claim 3, or a pharmaceutically acceptable salt or tautomer thereof, wherein R₁Is (C)₁-C₆) An alkyl group; r₂Is H; r₃And R₄Each independently selected from H or (C)₁-C₆) An alkyl group; r₆Is halogen; and n is 2.

13. The compound of claim 1 selected from

7-amino-3-methyl-N- (4- (piperazin-1-yl) phenethyl) thieno [2,3-b ] pyrazine-6-carboxamide,

7-amino-N- (3-chloro-4- (piperazin-1-yl) phenethyl) -3-methylthioeno [2,3-b ] pyrazine-6-carboxamide,

7-amino-N- (2, 5-difluoro-4-piperazin-1-yl) phenethyl) -3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

or a pharmaceutically acceptable salt or tautomer thereof.

14. The compound of claim 1 selected from

7-amino-N- (2, 5-difluoro-4-piperazin-1-yl) phenethyl) -3-methylthieno [2,3-b ] pyrazine-6-carboxamide, or a pharmaceutically acceptable salt or tautomer thereof.

15. A compound selected from

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl-3-chloroethyl-7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) phenethyl) -7-amino-3-methylthio-eno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenethyl) -7-amino-3-methylthio-eno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 5-difluorophenethyl) -7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-cyanophenylethyl) -7-amino-3-methylthio [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2-fluorophenethyl) -7-amino-3-methylthio-eno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 6-difluorophenethyl) -7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxamide,

N- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 5-difluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2, 5-difluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -2-fluorophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2S) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-cyanophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2R) -2- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) -3-cyanophenyl) propyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- (4- (9-oxa-3, 7-diazabicyclo [3.3.1] nonan-3-yl) -2, 5-difluorophenethyl) -7-amino-3-methylthieno [2,3-b ] pyrazine-6-carboxamide,

N- ((2R) -2- (4- (9-oxa-3, 7-diazabicyclo [3.3.1] nonan-3-yl) -2, 5-difluorophenyl) propane

Yl) -7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxamide, and

n- ((2S) -2- (4- (9-oxa-3, 7-diazabicyclo [3.3.1] nonan-3-yl) -2, 5-difluorophenyl) propyl) -7-amino-3-methylthioeno [2,3-b ] pyrazine-6-carboxamide,

or a pharmaceutically acceptable salt or tautomer thereof.

16. A compound which is:

n- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) phenethyl) -7-amino-2-ethylthieno [2,3-b ] pyrazine-6-carboxamide, or a pharmaceutically acceptable salt or tautomer thereof.

17. A compound which is:

n- (4- (3, 8-diazabicyclo [3.2.1] octan-3-yl) phenethyl) -7-amino-3-ethylthieno [2,3-b ] pyrazine-6-carboxamide, or a pharmaceutically acceptable salt or tautomer thereof.